U.S. patent application number 14/759386 was filed with the patent office on 2016-02-11 for deoxyuridine triphosphatase inhibitors.
The applicant listed for this patent is UNIVERSITY OF SOUTHERN CALIFORNIA. Invention is credited to Bruno Giethlen, Robert D. Ladner.
Application Number | 20160039788 14/759386 |
Document ID | / |
Family ID | 50030487 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160039788 |
Kind Code |
A1 |
Ladner; Robert D. ; et
al. |
February 11, 2016 |
DEOXYURIDINE TRIPHOSPHATASE INHIBITORS
Abstract
Provided herein are dUTPase inhibitors, compositions comprising
such compounds and methods of using such compounds and
compositions. ##STR00001##
Inventors: |
Ladner; Robert D.; (Santa
Monica, CA) ; Giethlen; Bruno; (Altorf, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF SOUTHERN CALIFORNIA |
Los Angeles |
CA |
US |
|
|
Family ID: |
50030487 |
Appl. No.: |
14/759386 |
Filed: |
January 3, 2014 |
PCT Filed: |
January 3, 2014 |
PCT NO: |
PCT/US2014/010247 |
371 Date: |
July 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61874643 |
Sep 6, 2013 |
|
|
|
61749791 |
Jan 7, 2013 |
|
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|
Current U.S.
Class: |
514/230.5 ;
435/184; 435/375; 514/274; 514/300; 514/312; 514/326; 514/328;
514/369; 544/105; 544/312; 546/113; 546/122; 546/158; 546/210;
546/219; 548/183 |
Current CPC
Class: |
C07D 277/36 20130101;
C07D 211/88 20130101; C07D 239/54 20130101; C07D 403/06 20130101;
C07D 277/34 20130101; C07D 265/18 20130101; C07D 401/06 20130101;
C07D 231/56 20130101; A61P 35/00 20180101; C07D 498/04 20130101;
C07D 401/12 20130101; C07D 215/26 20130101; C07D 471/04 20130101;
A61P 43/00 20180101; A61P 35/02 20180101 |
International
Class: |
C07D 401/06 20060101
C07D401/06; C07D 277/34 20060101 C07D277/34; C07D 239/54 20060101
C07D239/54; C07D 471/04 20060101 C07D471/04; C07D 498/04 20060101
C07D498/04; C07D 211/88 20060101 C07D211/88; C07D 401/12 20060101
C07D401/12 |
Claims
1. A compound of formula (I) or (II): ##STR00071## or a tautomer
thereof, including any stereoisomer, enantiomer or diastereoisomer,
or a pharmaceutically acceptable salt of each thereof, wherein
##STR00072## is a uracil isostere; W is a bond or optionally
substituted --CH.sub.2--; W.sup.1 is a bond, N, or an optionally
substituted CH group; X is a bond, O, S, NR.sup.19, optionally
substituted C.sub.1-C.sub.6 alkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, or optionally substituted
C.sub.2-C.sub.6 alkynylene group, a divalent optionally substituted
C.sub.6-C.sub.10 aromatic hydrocarbon group, or a divalent
optionally substituted saturated or unsaturated C.sub.2-C.sub.10
heterocyclic or optionally substituted C.sub.1-C.sub.10heteroaryl
group; R.sup.19 is hydrogen, optionally substituted C.sub.1-C.sub.6
alkyl or optionally substituted C.sub.3-C.sub.5 cycloalkyl; Y is a
bond or a linear or branched optionally substituted
C.sub.1-C.sub.10 alkylene which further optionally has a
cycloalkylidene structure on one carbon atom, or is optionally
substituted C.sub.2-C.sub.6 alkenylene, or optionally substituted
C.sub.2-C.sub.6 alkynylene group; Z is
--PO.sub.2--NR.sup.31R.sup.32, --SO.sub.2NR.sup.31R.sup.32,
--NR.sup.3PO.sub.2--R.sup.4, or --NR.sup.3SO.sub.2--R.sup.4,
wherein R.sup.31 and R.sup.32 are the same or different and each
represents a hydrogen atom, optionally substituted C.sub.1-C.sub.6
alkyl group optionally substituted with an aryl group, wherein the
aryl group, together with the R.sup.31 or R.sup.32, may form a
condensed bicyclic hydrocarbon, or R.sup.1 and R.sup.2 are taken
together with the adjacent nitrogen atom form an optionally
substituted C.sub.2-C.sub.10 heterocyclic group; Z.sup.1 is
--PO.sub.2--NR.sup.31R.sup.32 or R.sup.3PO.sub.2--R.sup.4 wherein
R.sup.31 and R.sup.32 are independently a hydrogen atom, optionally
substituted C.sub.1-C.sub.6 alkyl group optionally substituted with
an aryl group, wherein the aryl group, together with the R.sup.31
or R.sup.32, may form a condensed bicyclic hydrocarbon, or R.sup.31
and R.sup.32 are taken together with the adjacent nitrogen atom
form an optionally substituted C.sub.2-C.sub.10 heterocyclic group;
R.sup.3 is hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl; and R.sup.4 is optionally substituted C.sub.6-C.sub.10 aryl
or an optionally substituted C.sub.2-C.sub.10 heterocyclic
group.
2. The compound of claim 1, wherein the uracil isostere is an
optionally substituted cycloalkyl or optionally substituted
heterocyclyl ring which is monocyclic, bicyclic, tricyclic, or
tetracyclic, wherein the ring comprises a moiety selected from
--C(.dbd.V)--NH--C(.dbd.V)--, --C(.dbd.V)--CH.sub.2--C(.dbd.V)--,
optionally substituted meta-dihaho phenyl, such as meta-difluoro
phenyl, ##STR00073## wherein each V independently is O or S, each
R.sup.1 independently is hydrogen, C.sub.1-C.sub.6 alkyl optionally
substituted with C.sub.3-C.sub.5 cycloalkyl, or C.sub.3-C.sub.5
cycloalkyl, each R.sup.2 is independently --OH, --SH, --OR.sup.1,
--SR.sup.1, or halo wherein R.sup.1 is defined as above, R.sup.10
is hydrogen, R.sup.12, or --O--R.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl optionally substituted with 1-3 hydroxy, fluoro, chloro,
and amino substituent, R.sup.11 is hydrogen, halo, R.sup.12 or
--O--R.sup.12, wherein R.sup.12 is defined as above, r is 1, 2, or
3, each Q.sup.1 and Q.sup.2 independently are --CH.sub.2--, O, S or
an oxidized form thereof, NH or an oxidized form thereof, or
Q.sup.1 and Q.sup.2 together form a --CH.dbd.CH-- moiety; provided
that Q.sup.1 and Q.sup.2 are both not O, S or an oxidized form
thereof, NH or an oxidized form thereof or a combination of each
thereof; wherein each --CH.dbd., --CH.sub.2--, and --NH-- is
optionally substituted.
3-15. (canceled)
16. The compound of claim 1 wherein the uracil isostere is:
##STR00074##
17. The compound of claim 1, wherein the uracil isostere is:
##STR00075##
18. The compound of claim 1 wherein the uracil isostere is:
##STR00076##
19. The compound of claim 1 wherein the uracil isostere is:
##STR00077##
20. The compound of claim 1 wherein the uracil isostere is:
##STR00078##
21. The compound of claim 1, wherein --W--X--Y-- is
--CH.sub.2--X--SO.sub.2--NH--CH(R.sup.Y)--,
--CH.sub.2--X--SO.sub.2--NH--C(R.sup.Y).sub.2--, or
--CH.sub.2--X--B--CH.sub.2CR.sup.ZR.sup.W--X is optionally
substituted C.sub.1-C.sub.6 alkylene wherein one of the methylene
groups within the alkylene chain is optionally replaced with an O
or S atom, such that X is optionally substituted alkylene or a
heteroalkylene; B is a optionally substituted C.sub.3-C.sub.10
heteroaryl; R.sup.Y an R.sup.w are independently hydrogen or
optionally substituted C.sub.1-C.sub.6 alkyl; and R.sup.z is
hydrogen or hydroxy.
22. The compound of claim 1, wherein --W--X--Y-- is ##STR00079##
wherein Y.sup.1 is CH.sub.2, O or S, X.sup.10 is NH,
NCO.sub.2R.sup.20, O, or CH.sub.2, R.sup.20 is C.sub.1-C.sub.6
alkyl optionally substituted with 1-3 C.sub.6-C.sub.10 aryl groups,
u is 0, 1, 2, 3, or 4, and R.sup.z is hydroxy or hydrogen, and
R.sup.w is C.sub.1-C.sub.6 alkyl or hydrogen, and the phenylene and
the heteroarylene rings are optionally substituted.
23. The compound of claim 1, wherein Z is: ##STR00080## R.sup.6 is
hydrogen or halo, and R.sup.7 is optionally substituted
C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.2-C.sub.6
alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally
substituted C.sub.3-C.sub.5 cycloalkyl, optionally substituted
C.sub.3-C.sub.10 heteroaryl, optionally substituted
C.sub.3-C.sub.10 heterocyclyl, or optionally substituted
phenyl.
24. A compound, or a tautomer thereof, including any stereoisomer,
enantiomer or diastereoisomer, and pharmaceutically acceptable salt
of each thereof, wherein the compound selected from: ##STR00081##
##STR00082## ##STR00083##
25. A compound, or a tautomer thereof, including any stereoisomer,
enantiomer or diastereoisomer, or a pharmaceutically acceptable
salt of each thereof of formula (III): ##STR00084## wherein A is
##STR00085## R.sup.10 is hydrogen, R.sup.12, or --O--R.sup.12,
R.sup.12 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.6 alkynyl optionally substituted with 1-3 hydroxy,
fluoro, chloro, and amino substituent, R.sup.11 is hydrogen, halo,
R.sup.12 or --O--R.sup.12, wherein R.sup.12 is defined as above, r
is 1, 2, or 3, L.sup.1- is ##STR00086## wherein Y.sup.1 is
CH.sub.2, O, S, X.sup.10 is NH, NCO.sub.2R.sup.20, O, or CH.sub.2,
R.sup.20 is C.sub.1-C.sub.6 alkyl optionally substituted with 1-3
C.sub.6-C.sub.10aryl groups, u is 0, 1, 2, 3, or 4, R.sup.z is
hydroxy or hydrogen, R.sup.w is C.sub.1-C.sub.6 alkyl or hydrogen,
and the phenylene and the heteroarylene rings are optionally
substituted, Z is phenyl or a 5 or 6 member heteroaryl substituted
with an R.sup.6 and an R.sup.60 groups, wherein the R.sup.6 and the
R.sup.6 are positioned 1,2 with respect to each other, R.sup.6 is
hydrogen, optionally substituted C.sub.1-C.sub.6 alkoxy, or halo,
and R.sup.60 is --OR.sup.7 or --NHR.sup.7R.sup.70, R.sup.7 is
optionally substituted C.sub.1-C.sub.10 alkyl, optionally
substituted C.sub.2-C.sub.6 alkenyl, optionally substituted
C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.3-C.sub.5
cycloalkyl, optionally substituted C.sub.3-C.sub.10 heteroaryl,
optionally substituted C.sub.3-C.sub.10 heterocyclyl, or optionally
substituted phenyl, and R.sup.70 is hydrogen or R.sup.7.
26. The compound of claim 25, wherein L.sup.1 is ##STR00087##
27. The compound of claim 25, wherein L.sup.1 is ##STR00088##
28. The compound of claim 25, wherein the uracil isostere is:
##STR00089##
29. The compound of claim 25, wherein the uracil isostere is:
##STR00090##
30. The compound of claim 25, wherein the uracil isostere is:
##STR00091##
31. The compound of claim 25, wherein the uracil isostere is:
##STR00092##
32. The compound of claim 25, wherein Z is selected from:
##STR00093## wherein each R.sup.6 and R.sup.7 independently are
defined as in claim 25 above, each R.sup.61 and R.sup.62
independently is N or CH, provided that at least one of R.sup.61
and R.sup.62 is N, each R.sup.63 independently is NR.sup.70, S, O,
and each R.sup.64 independently is N or CH.
33. The compound of claim 25 of formula: ##STR00094##
34. The compound of claim 25, of wherein L.sup.1 is defined as in
claim 24 above, R.sup.6 is hydrogen, F, Cl, OMe, or OCF.sub.3, and
R.sup.7 is ##STR00095## wherein t is 1, 2, or 3.
35. A stereochemically pure enantiomer of a compound of claim 1, or
its pharmaceutically acceptable salt.
36. Compound PCI 10586 or pharmaceutically acceptable salt
thereof.
37. A composition comprising the compound of claim 1 and a carrier
or an excipient.
38. The composition of claim 37, wherein the carrier or the
excipient is a pharmaceutically acceptable carrier or
excipient.
39. A method of one or more of inhibiting dUTPase or enhancing the
efficacy of a dUTPase directed therapy comprising contacting the
dUTPase with the compound of claim 1.
40-52. (canceled)
53. A method of treating a disease whose treatment is impeded by
the expression or over expression of dUTPase, comprising
administering to a patient in need of such treatment an effective
amount of the compound of claim 1.
54-61. (canceled)
62. A method of inhibiting the growth of a cancer cell comprising
contacting the cell with an effective amount of the compound of
claim 1 and an effective amount of a dUTPase-directed therapeutic,
thereby inhibiting the growth of the cancer cell.
63-75. (canceled)
76. A method of treating a disease in a patient whose treatment is
impeded by the expression or over expression of dUTPase,
comprising: a. screening a cell or tissue sample isolated from the
patient; b. determining the expression level of dUTPase in the
sample, c. administering to a patient whose sample shows over
expression of dUTPase an effective amount of the compound of claim
1.
77-78. (canceled)
79. The compound of claim 1 of formula: ##STR00096## wherein A is
selected from: X.sup.10 is NH, NCO.sub.2R.sup.20, O, or CH.sub.2;
R.sup.20 is C.sub.1-C.sub.6 alkyl optionally substituted with 1-3
C.sub.6-C.sub.10 aryl groups; u is 0, 1, 2, 3, or 4; R.sup.11 is
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.6 alkynyl wherein each alkyl, alkenyl, and alkynyl is
optionally substituted with 1-3 hydroxy, fluoro, chloro, and amino
substituent; R.sub.60 is C.sub.1-C.sub.6 alkyl, and r is 1, 2, or
3.
80. The compound of claim 79, wherein A is: ##STR00097##
81. The compound of claim 80, wherein A is selected from:
##STR00098##
82-85. (canceled)
86. The compound of claim 79, selected from: ##STR00099##
##STR00100## and a diastereomer or an enantiomer thereof, or a
pharmaceutically acceptable salt thereof.
87. A composition comprising the compound of claim 79.
88. The composition of claim 87, wherein the carrier or the
excipient is a pharmaceutically acceptable carrier or
excipient.
89. A method of one or more of inhibiting dUTPase or enhancing the
efficacy of a dUTPase directed therapy comprising contacting the
dUTPase with the compound of claim 79.
90-128. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. section
119(e) to U.S. Provisional Application Ser. Nos. 61/749,791 filed
Jan. 7, 2013, and 61/874,643 filed Sep. 6, 2013, the content of
each of which is incorporated herein in its entirety by
reference.
BACKGROUND
[0002] Thymidylate metabolism is required for producing essential
building blocks necessary to replicate DNA in dividing cells and
has long been an important therapeutic target for cornerstone
cancer drugs. Drugs targeting this pathway such as 5-fluorouracil
(5-FU) inhibit the enzyme thymidylate synthase (TS) and are
currently critical standard-of care therapies. TS-targeted agents
are heavily used for the treatment of a variety of cancers
including colon, gastric, head and neck, breast, lung and blood
related malignancies among others. Grem, J. L., 5-Fluorouracil plus
leucovorin in cancer therapy, in Principals and Practice of
Oncology Update Series, J. De Vita, V. T., S. Hellman, and A.
Rosenberg, Editors. 1988, J. B. Lippincott: Philadelphia, Pa.
[0003] There are two classes of drugs that target the TS enzyme:
the fluoropyrimidines and the antifolates. The fluoropyrimidines, 5
FU, S-1 and capecitabine (Xeloda.RTM.), have wide use in the
treatment of gastrointestinal and breast cancers, while the
antifolate pemetrexed (Alimt.RTM.) is currently used for the
treatment of non-small cell lung cancer (NSCLC). Since the
discovery of 5-FU over fifty years ago by Charles Heidelberger, the
fluoropyrimidines remain one of the most common and effective
anticancer cancer drugs used worldwide. Due to this fact, there is
an abundance of clinical experience and insight into the mechanism
of action of these agents.
[0004] The TS inhibitor 5-fluorouracil (5 FU) remains the
foundation of many first and second line regimens in the treatment
of colon cancer. Single agent therapies including oxaliplatin,
irinotecan, Erbitux and Avastin, demonstrate lowered activity in
colon cancer as compared to 5-FU. In addition to colon cancer,
TS-directed agents have demonstrated efficacy in several other
solid tumor types.
[0005] Deoxyuridine triphosphatase ("dUTPase") is a ubiquitous
enzyme that is essential for viability in both prokaryotic and
eukaryotic organisms; as the main regulator of dUTP pools, the
expression of dUTPase could have profound effects on the utility of
chemotherapeutics that inhibit thymidylate biosynthesis. Normally,
dUTPase mediates a protective role by limiting the expansion of
dUTP pools and countering the cytotoxic effect of uracil
misincorporation. According to this model, elevated levels of
dUTPase could prevent TS inhibitor-induced dUTP accumulation and
induce drug resistance. It has been shown that dUTPase over
expression results in a significant decrease in dUTP accumulation
and increased resistance to drug treatment when compared to
controls.
[0006] Chemotherapeutic agents that target de novo thymidylate
metabolism are critical for the treatment of a variety of solid
tumors, however clinical efficacy is often hindered by drug
resistance. Because resistance to these agents is a common
occurrence, the identification and exploitation of novel
determinants of drug sensitivity within this pathway of proven
therapeutic utility is important. As disclosed by Ladner et al. in
U.S. Patent Publ. No. US 2011/0212467 ("Ladner"), uracil-DNA
misincorporation pathway can play a driving role in mediating
cytotoxicity to TS-directed chemotherapies.
[0007] For example, nearly half of cancer patients do not benefit
from 5-FU-based treatment due to intrinsic or acquired drug
resistance. Due to this fact, there is a critical need to overcome
the fundamental challenge of drug resistance and provide new
therapeutic strategies to improve patient outcome. This disclosure
satisfies this need and provides related advantages as well.
SUMMARY
[0008] In some aspects, this disclosure provides compounds,
compositions and methods that inhibit dUTPase when used alone or in
combination with at least one dUTPase-directed chemotherapy. In
some aspects, this disclosure provides compounds, compositions and
methods for treating cancer, killing cancer cells, and/or
inhibiting cancer cell growth when used in combination with at
least one TS-directed chemotherapy. Compounds of this class include
the following compounds of formulas (I), (II), and (III).
[0009] Thus, in one aspect, provided herein are compounds of
formulas (I), (II), and (III):
##STR00002##
or a tautomer thereof, or a pharmaceutically acceptable salt of
each thereof, wherein
##STR00003##
is a uracil isostere or a halo uracil;
##STR00004##
is uracil, halo uracil, or a uracil isostere; W is a bond or
optionally substituted --CH.sub.2--; W.sup.1 is a bond, N, or an
optionally substituted CH group; X is a bond, O, S, NR.sup.19,
optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.2-C.sub.6 alkenylene, or optionally substituted
C.sub.2-C.sub.6 alkynylene group, a divalent optionally substituted
C.sub.6-C.sub.10 aromatic hydrocarbon group, or a divalent
optionally substituted saturated or unsaturated C.sub.2-C.sub.10
heterocyclic or optionally substituted C.sub.1-C.sub.10 heteroaryl
group; R.sup.19 is hydrogen, optionally substituted C.sub.1-C.sub.6
alkyl or optionally substituted C.sub.3-C.sub.8 cycloalkyl; Y is a
bond or an optionally substituted C.sub.1-C.sub.10 alkylene which
further optionally has a cycloalkylidene structure on one carbon
atom, or is optionally substituted C.sub.2-C.sub.6 alkenylene, or
optionally substituted C.sub.2-C.sub.6 alkynylene group, or Y is
-L.sup.10-B.sup.1-L.sup.11-; L.sup.10 and L.sup.11 independently
are optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.2-C.sub.6 alkenylene, or optionally substituted
C.sub.2-C.sub.6 alkynylene group; B.sup.1 is a divalent optionally
substituted C.sub.6-C.sub.10 aromatic hydrocarbon group, or a
divalent optionally substituted saturated or unsaturated
C.sub.2-C.sub.10 heterocyclic or optionally substituted
C.sub.1-C.sub.10 heteroaryl group; Z is
--PO.sub.2--NR.sup.31R.sup.32, --SO.sub.2NR.sup.31R.sup.32,
--NR.sup.3PO.sub.2--R.sup.4, --NR.sup.3SO.sub.2--R.sup.4, or
R.sup.4 wherein R.sup.31 and R.sup.32 are the same or different and
each represents a hydrogen atom, optionally substituted
C.sub.1-C.sub.6 alkyl group optionally substituted with an aryl
group, wherein the aryl group, together with the R.sub.1 or
R.sub.2, may form a condensed bicyclic hydrocarbon, or R.sup.31 and
R.sup.32 are taken together with the adjacent nitrogen atom form an
optionally substituted C.sub.2-C.sub.10 heterocyclic group or an
optionally substituted C.sub.1-C.sub.10 heteroaryl group; Z.sup.1
is --PO.sub.2--NR.sup.31R.sup.32 or --(OR.sup.3)P(O)--R.sup.4
wherein R.sup.31 and R.sup.32 are independently a hydrogen atom,
optionally substituted C.sub.1-C.sub.6 alkyl group optionally
substituted with an aryl group, wherein the aryl group, together
with the R.sup.31 or R.sup.32, may form a condensed bicyclic
hydrocarbon, or R.sup.31 and R.sup.32 taken together with the
adjacent nitrogen atom form an optionally substituted
C.sub.2-C.sub.10 heterocyclic group or an optionally substituted
C.sub.1-C.sub.10 heteroaryl group; R.sup.3 is hydrogen or
optionally substituted C.sub.1-C.sub.6 alkyl; and R.sup.4 is
optionally substituted C.sub.6-C.sub.10 aryl, an optionally
substituted C.sub.2-C.sub.10 heterocyclic group, or an optionally
substituted C.sub.6-C.sub.10 heteroaryl group.
[0010] This disclosure also provides a tautotomer, or its
pharmaceutically acceptable salt of a compound as disclosed herein.
Methods to prepare such are known in the art.
[0011] This disclosure also provides a stereochemically pure
enantiomer of a compound as described herein, its tautotomer,
diastereoisomer or its pharmaceutically acceptable salt. Methods to
purify and identify the pure enantiomer are known in the art and
described herein.
[0012] In one aspect, the compound is provided as a
stereochemically pure enantiomer, e.g., PCI 10586, as described
herein. Pharmaceutically acceptable salts of PCI 10586 are also
provided herein.
[0013] In another aspect, compositions comprising one or more of
the above-noted compounds and a carrier are provided. In one
embodiment, the composition is a pharmaceutical composition and
therefore further comprises at least a pharmaceutically acceptable
carrier or a pharmaceutically acceptable excipient. The
compositions are formulated for various delivery modes, e.g.,
systemic (oral) or local.
[0014] In another aspect, this disclosure provides compositions
comprising one or more compounds as provided herein and a
dUTPase-directed chemotherapy and a carrier, such as a
pharmaceutically acceptable carrier. The compound and chemotherapy
can be in varying amounts, and in one aspect, each in an effective
amount when used in combination, provides a therapeutic benefit as
described herein. The compositions are formulated for various
delivery modes, e.g., systemic (oral) or local.
[0015] In another aspect, methods are provided for inhibiting
deoxyuridine triphosphatase (dUTPase) comprising contacting the
dUTPase with an effective amount of a compound or a composition
provided herein. In another aspect, the method further comprises
contacting the dUTPase with a dUTPase-directed chemotherapy alone
or in combination with the compound as provided herein. The
contacting can be simultaneous or concurrent. In a further aspect
the dUTPase-directed chemotherapy is contacted prior to the
compound or composition as described herein. In another aspect, the
dUTPase-directed chemotherapy is contacted subsequent to the
compound or composition. In a yet further aspect, the compound or
composition and the dUTPase-directed chemotherapy are sequentially
administered through several rounds of therapy. The contacting can
be simultaneous or concurrent and/or in vitro (cell free), ex vivo
or in vivo. In a further aspect, the compounds or compositions of
this disclosure are administered to a patient identified or
selected for the therapy by determining that the patient has a
tumor or mass that over expresses dUTPase. Methods to identify such
patients are known in the art and incorporated herein. The methods
when administered to a subject such as a human patient, can be
first line, second line, third line, forth line or further
therapy.
[0016] Also provided is a method for reversing resistance to a
dUTPase-directed chemotherapy comprising contacting a cell over
expressing dUTPase with an effective amount of a compound or a
composition provided herein, alone or in combination with a
dUTPase-directed chemotherapy. In one aspect, the cell is first
identified as over expressing dUTPase by a screen as disclosed by
U.S. Pat. No. 5,962,246. In another aspect, the method further
comprises subsequently contacting the cell expressing dUTPase with
a dUTPase-directed chemotherapy. The methods can be administered as
second line, third line, forth line or further therapy.
[0017] Further provided is a method for enhancing the efficacy of a
dUTPase-directed chemotherapy comprising contacting a cell, e.g.,
in one aspect a over expressing dUTPase, with an effective amount
of a compound or a composition provided herein. In another aspect,
the method further comprises contacting the cell with a
dUTPase-directed chemotherapy. The contacting can be simultaneous
or concurrent and/or in vitro (cell free), ex vivo or in vivo. In a
further aspect the dUTPase-directed chemotherapy is contacted prior
to the compound or composition as described herein, or vice versa.
The methods when administered to a subject such as a human patient,
can be first line, second line, third line, forth line or further
therapy.
[0018] In another aspect, provided herein is a method of treating a
disease associated with the dUTPase pathway, e.g., cancer, viral
infection, bacterial infection, or an autoimmune disorder,
comprising administering to a patient in need of such treatment an
effective amount of the compound provided herein or a composition
provided herein in combination with an agent which is suitable for
treating the disease, thereby treating the disease. The
administration of the compound of this invention and the agent that
is suitable for the disease (e.g., a dUTPase inhibitor) can be
simultaneous or concurrent and/or in vitro (cell free), ex vivo or
in vivo. In a further aspect the agent that is suitable for
treating the disease is administered prior to the compound or
composition as described herein, or vice versa. In one aspect, the
patient being treated is selected for the therapy by screening a
cell or tissue sample isolated from the patient for over expression
of dUTPase. The therapy is then administered to this patient after
the screen.
[0019] In another aspect, provided herein is a kit comprising a
compound provided herein or a composition provided herein and one
more of a dUTPase inhibitor (e.g., an antitumor agent) and
instructions for administering the agent. Yet further provided in
the kit are reagents and instructions to screen for dUTPase
expression.
[0020] In each of the above embodiments, a non-limiting example of
the dUTPase mediated chemotherapy comprises a TS-inhibitor, e.g.,
5-FU or 5-FU containing therapy such as 5-FU based adjuvant therapy
and chemical equivalents thereof.
BRIEF DESCRIPTION OF FIGURES
[0021] FIGS. 1A and B show characterization of PCI 10213 by (A)
HPLC (A1) and MS (A2) and (B) .sup.1H-NMR.
[0022] FIGS. 2A and B show characterization of PCI 10214 by (A)
HPLC (A1) and MS (A2) (B) .sup.1H-NMR.
[0023] FIGS. 3A-C show in (A) dUTPase enzyme inhibition assay
showing % inhibition at increasing concentrations of PCI 10213 and
PCI 10216, and in (B) and (C), MTS assays where colon cancer and
NSCLC cancer cells were treated with PCI 10213, PCI 10214 and PCI
10216 alone. Data is presented as % control of vehicle-treated
controls.
[0024] FIGS. 4A and B show MTS assay where HCT116 (A) and SW620 (B)
colon cancer cells were treated with a fixed dose of 25 .mu.mol/L
PCI 10213 or PCI 10216 alone and in combination with increasing
doses of 5-FU. Data is presented as % control of vehicle-treated
controls.
[0025] FIG. 5 shows a colony formation assay, where NSCLC, colon
and breast cancer cells were treated with PCI 10213 alone and in
combination with a fixed dose of FUdR. Data is presented as %
control of vehicle-treated controls. Bars represent mean.+-.SEM.
Representative images for one NSCLC, one colon and one breast
cancer cell line are showing in FIGS. 6 and 7.
[0026] FIGS. 6A-6D show a colony formation assay, where HCT116
colon cancer cells were treated with PCI 10213, PCI 10214, PCI
10216 alone and in combination with a fixed dose of FUdR.
Representative images are scans of the colonies stained with
crystal violet. (A) Cells treated with increasing concentrations of
FUdR alone. (B) Cells treated with increasing concentrations of PCI
10213 alone (top row) and combination with 0.5 .mu.mol/L FUdR. (C)
Cells treated with increasing concentrations of PCI 10214 alone
(top row) and combination with 0.5 .mu.mol/L FUdR. (D) Cells
treated with increasing concentrations of PCI 10216 alone (top row)
and combination with 0.5 .mu.mol/L FUdR.
[0027] FIGS. 7A-C show colony formation assay, where A549 NSCLC
cells were treated with PCI 10213 or PCI 10216 alone and in
combination with a fixed dose of FUdR. Representative images are
scans of the colonies stained with crystal violet. (A) Cells
treated with increasing concentrations of FUdR alone. (B) Cells
treated with increasing concentrations of PCI 10213 alone (top row)
and combination with 0.5 .mu.mol/L FUdR. (C) Cells treated with
increasing concentrations of PCI 10216 alone (top row) and
combination with 0.5 .mu.mol/L FUdR.
[0028] FIGS. 8A-C show a colony formation assay, where MCF7 breast
cancer cells were treated with PCI 10213 or PCI 10216 alone and in
combination with a fixed dose of FUdR. Representative images are
scans of the colonies stained with crystal violet. (A) Cells
treated with increasing concentrations of FUdR alone. (B) Cells
treated with increasing concentrations of PCI 10213 alone (top row)
and combination with 0.5 .mu.mol/L FUdR. (C) Cells treated with
increasing concentrations of PCI 10216 alone (top row) and
combination with 0.5 .mu.mol/L FUdR.
[0029] FIG. 9 shows HPLC chromatogram of PCI 10586 with retention
time (R.sub.t)=28.4 min.
[0030] FIG. 10 shows HPLC chromatogram of PCI 10585 with
R.sub.t=22.13 min.
[0031] FIGS. 11A-11C show the results of a colony forming assay.
HCT116 colon cancer cells were treated with PCI 10213, PCI 10585,
PCI 102586 alone and in combination with a fixed dose of FUdR.
Representative images are scans of the colonies stained with
crystal violet. (A) Cells treated with increasing concentrations of
FUdR alone. (B) Cells treated with increasing concentrations of PCI
10213, 10585, 10586 alone. (C) Cells treated with increasing
concentrations of PCI 10213, 10585 and 10586 in combination with
0.5 .mu.mol/L FUdR.
[0032] FIG. 12 graphically shows quantitation of a colony formation
assay. Briefly, HCT116 colon cancer cells were treated with PCI
10213, PCI 10585, PCI 102586 alone and in combination with a fixed
dose of 0.5 .mu.mol/L FUdR. Bars represent the number of colonies
counted following staining with crystal violet. Top, PCI 10213;
middle, PCI 10585; bottom, PCI 10586.
DETAILED DESCRIPTION
[0033] Throughout this disclosure, various publications, patents
and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure in their entirety to more
fully describe the state of the art to which this invention
pertains.
DEFINITIONS
[0034] The practice of the present technology will employ, unless
otherwise indicated, conventional techniques of organic chemistry,
pharmacology, immunology, molecular biology, microbiology, cell
biology and recombinant DNA, which are within the skill of the art.
See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A
Laboratory Manual, 2.sup.nd edition (1989); Current Protocols In
Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series
Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical
Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory
Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)).
[0035] As used in the specification and claims, the singular form
"a," "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0036] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
not exclude others. "Consisting essentially of" when used to define
compositions and methods, shall mean excluding other elements of
any essential significance to the combination. Thus, a composition
consisting essentially of the elements as defined herein would not
exclude trace contaminants, e.g., from the isolation and
purification method and pharmaceutically acceptable carriers,
preservatives, and the like. "Consisting of" shall mean excluding
more than trace elements of other ingredients. Embodiments defined
by each of these transition terms are within the scope of this
technology.
[0037] All numerical designations, e.g., pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 1, 5,
or 10%. It is to be understood, although not always explicitly
stated that all numerical designations are preceded by the term
"about." It also is to be understood, although not always
explicitly stated, that the reagents described herein are merely
exemplary and that equivalents of such are known in the art.
[0038] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and preferably 1 to 6
carbon atoms. This term includes, by way of example, linear and
branched hydrocarbyl groups such as methyl (CH.sub.3--), ethyl
(CH.sub.3CH.sub.2--), n-propyl (CH.sub.3CH.sub.2CH.sub.2--),
isopropyl ((CH.sub.3).sub.2CH--), n-butyl
(CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), isobutyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neopentyl ((CH.sub.3).sub.3CCH.sub.2--).
[0039] "Alkenyl" refers to monovalent straight or branched
hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2
to 4 carbon atoms and having at least 1 and preferably from 1 to 2
sites of vinyl (>C.dbd.C<) unsaturation. Such groups are
exemplified, for example, by vinyl, allyl, and but-3-en-1-yl.
Included within this term are the cis and trans isomers or mixtures
of these isomers.
[0040] "Alkynyl" refers to straight or branched monovalent
hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2
to 3 carbon atoms and having at least 1 and preferably from 1 to 2
sites of acetylenic (--C.ident.C--) unsaturation. Examples of such
alkynyl groups include acetylenyl (--C.dbd.CH), and propargyl
(--CH.sub.2C.ident.CH).
[0041] "Substituted alkyl" refers to an alkyl group having from 1
to 5, preferably 1 to 3, or more preferably 1 to 2 substituents
selected from the group consisting of alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
substituted sulfonyloxy, thioacyl, thiol, alkylthio, and
substituted alkylthio, wherein said substituents are as defined
herein.
[0042] "Substituted alkenyl" refers to alkenyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxyl,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
substituted sulfonyloxy, thioacyl, thiol, alkylthio, and
substituted alkylthio, wherein said substituents are as defined
herein and with the proviso that any hydroxyl or thiol substitution
is not attached to a vinyl (unsaturated) carbon atom.
[0043] "Substituted alkynyl" refers to alkynyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
substituted sulfonyloxy, thioacyl, thiol, alkylthio, and
substituted alkylthio, wherein said substituents are as defined
herein and with the proviso that any hydroxyl or thiol substitution
is not attached to an acetylenic carbon atom.
[0044] "Alkylene" refers to divalent saturated aliphatic
hydrocarbyl groups preferably having from 1 to 6 and more
preferably 1 to 3 carbon atoms that are either straight-chained or
branched. This term is exemplified by groups such as methylene
(--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--), n-propylene
(--CH.sub.2CH.sub.2CH.sub.2--), iso-propylene
(--CH.sub.2CH(CH.sub.3)-- or --CH(CH.sub.3)CH.sub.2--), butylene
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), isobutylene
(--CH.sub.2CH(CH.sub.3)CH.sub.2--), sec-butylene
(--CH.sub.2CH.sub.2(CH.sub.3)CH--), and the like. Similarly,
"alkenylene" and "alkynylene" refer to an alkylene moiety
containing respective 1 or 2 carbon carbon double bonds or a carbon
carbon triple bond.
[0045] "Substituted alkylene" refers to an alkylene group having
from 1 to 3 hydrogens replaced with substituents selected from the
group consisting of alkyl, substituted alkyl, alkoxy, substituted
alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,
aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy,
cyano, halogen, hydroxyl, nitro, carboxyl, carboxyl ester,
cycloalkyl, substituted cycloalkyl, heteroaryl, substituted
heteroaryl, heterocyclic, substituted heterocyclic, and oxo wherein
said substituents are defined herein.
[0046] In some embodiments, the alkylene has 1 to 2 of the
aforementioned groups, or having from 1-3 carbon atoms replaced
with --O--, --S--, or --NR.sup.Q-- moieties where R.sup.Q is H or
C.sub.1-C.sub.6 alkyl. It is to be noted that when the alkylene is
substituted by an oxo group, 2 hydrogens attached to the same
carbon of the alkylene group are replaced by ".dbd.O". "Substituted
alkenylene"and" substituted alkynylene" refer to alkenylene and
substituted alkynylene moieties substituted with substituents as
described for substituted alkylene.
[0047] "Alkoxy" refers to the group --O-alkyl wherein alkyl is
defined herein. Alkoxy includes, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and
n-pentoxy.
[0048] "Substituted alkoxy" refers to the group --O-(substituted
alkyl) wherein substituted alkyl is defined herein.
[0049] "Acyl" refers to the groups H--C(O)--, alkyl-C(O)--,
substituted alkyl-C(O)--, alkenyl-C(O)--, substituted
alkenyl-C(O)--, alkynyl-C(O)--, substituted alkynyl-C(O)--,
cycloalkyl-C(O)--, substituted cycloalkyl-C(O)--,
cycloalkenyl-C(O)--, substituted cycloalkenyl-C(O)--, aryl-C(O)--,
substituted aryl-C(O)--, heteroaryl-C(O)--, substituted
heteroaryl-C(O)--, heterocyclic-C(O)--, and substituted
heterocyclic-C(O)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined herein.
Acyl includes the "acetyl" group CH.sub.3C(O)--.
[0050] "Acylamino" refers to the groups --NR.sup.47C(O)alkyl,
--NR.sup.47C(O) substituted alkyl, --NR.sup.47C(O)cycloalkyl,
--NR.sup.47C(O) substituted cycloalkyl,
--NR.sup.47C(O)cycloalkenyl, --NR.sup.47C(O) substituted
cycloalkenyl, --NR.sup.47C(O)alkenyl, --NR.sup.47C(O) substituted
alkenyl, --NR.sup.47C(O)alkynyl, --NR.sup.47C(O) substituted
alkynyl, --NR.sup.47C(O)aryl, --NR.sup.47C(O) substituted aryl,
--NR.sup.47C(O)heteroaryl, --NR.sup.47C(O) substituted heteroaryl,
--NR.sup.47C(O)heterocyclic, and --NR.sup.47C(O) substituted
heterocyclic wherein R.sup.47 is hydrogen or alkyl and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0051] "Acyloxy" refers to the groups alkyl-C(O)O--, substituted
alkyl-C(O)O--, alkenyl-C(O)O--, substituted alkenyl-C(O)O--,
alkynyl-C(O)O--, substituted alkynyl-C(O)O--, aryl-C(O)O--,
substituted aryl-C(O)O--, cycloalkyl-C(O)O--, substituted
cycloalkyl-C(O)O--, cycloalkenyl-C(O)O--, substituted
cycloalkenyl-C(O)O--, heteroaryl-C(O)O--, substituted
heteroaryl-C(O)O--, heterocyclic-C(O)O--, and substituted
heterocyclic-C(O)O-- wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0052] An animal, subject or patient for diagnosis or treatment
refers to an animal such as a mammal, or a human, ovine, bovine,
feline, canine, equine, simian, etc. Non-human animals subject to
diagnosis or treatment include, for example, simians, murine, such
as, rat, mice, canine, leporid, livestock, sport animals, and
pets.
[0053] "Amino" refers to the group --NH.sub.2.
[0054] "Substituted amino" refers to the group --NR.sup.48R.sup.49
where R.sup.48 and R.sup.49 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, substituted heterocyclic, --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-alkenyl,
--SO.sub.2-substituted alkenyl, --SO.sub.2-cycloalkyl,
--SO.sub.2-substituted cycloalkyl, --SO.sub.2-cycloalkenyl,
--SO.sub.2-substituted cylcoalkenyl, --SO.sub.2-aryl,
--SO.sub.2-substituted aryl, --SO.sub.2-heteroaryl,
--SO.sub.2-substituted heteroaryl, --SO.sub.2-heterocyclic, and
--SO.sub.2-substituted heterocyclic and wherein R.sup.48 and
R.sup.49 are optionally joined, together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
provided that R.sup.48 and R.sup.49 are both not hydrogen, and
wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein. When R.sup.48 is hydrogen and
R.sup.49 is alkyl, the substituted amino group is sometimes
referred to herein as alkylamino. When R.sup.48 and R.sup.49 are
alkyl, the substituted amino group is sometimes referred to herein
as dialkylamino. When referring to a monosubstituted amino, it is
meant that either R.sup.48 or R.sup.49 is hydrogen but not both.
When referring to a disubstituted amino, it is meant that neither
R.sup.48 nor R.sup.49 are hydrogen.
[0055] "Aminocarbonyl" refers to the group --C(O)NR.sup.50R.sup.5'
where R.sup.50 and R.sup.51 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic and where R.sup.50 and
R.sup.51 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0056] "Aminothiocarbonyl" refers to the group
--C(S)NR.sup.50R.sup.51 where R.sup.50 and R.sup.51 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0057] "Aminocarbonylamino" refers to the group
--NR.sup.47C(O)NR.sup.50R.sup.51 where R.sup.47 is hydrogen or
alkyl and R.sup.50 and R.sup.51 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic, and where R.sup.50 and
R.sup.51 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0058] "Aminothiocarbonylamino" refers to the group
--NR.sup.47C(S)NR.sup.50R.sup.51 where R.sup.47 is hydrogen or
alkyl and R.sup.50 and R.sup.51 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic and where R.sup.50 and
R.sup.51 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0059] "Aminocarbonyloxy" refers to the group
--O--C(O)NR.sup.50R.sup.51 where R.sup.50 and R.sup.51 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0060] "Aminosulfonyl" refers to the group
--SO.sub.2NR.sup.50R.sup.51 where R.sup.50 and R.sup.51 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0061] "Aminosulfonyloxy" refers to the group
--O--SO.sub.2NR.sup.50R.sup.51 where R.sup.50 and R.sup.51 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0062] "Aminosulfonylamino" refers to the group
--NR.sup.47SO.sub.2NR.sup.50R.sup.51 where R.sup.47 is hydrogen or
alkyl and R.sup.50 and R.sup.51 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic and where R.sup.50 and
R.sup.51 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0063] "Amidino" refers to the group
--C(.dbd.NR.sup.52)NR.sup.50R.sup.51 where R.sup.50, R.sup.51, and
R.sup.52 are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.50 and R.sup.51 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0064] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic
group of from 6 to 14 carbon atoms having a single ring (e.g.,
phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)
which condensed rings may or may not be aromatic (e.g.,
2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like)
provided that the point of attachment is at an aromatic carbon
atom. Preferred aryl groups include phenyl and naphthyl.
[0065] "Substituted aryl" refers to aryl groups which are
substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to
2 substituents selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
substituted sulfonyloxy, thioacyl, thiol, alkylthio, and
substituted alkylthio, wherein said substituents are as defined
herein.
[0066] "Aryloxy" refers to the group --O-aryl, where aryl is as
defined herein, that includes, by way of example, phenoxy and
naphthoxy.
[0067] "Substituted aryloxy" refers to the group --O-(substituted
aryl) where substituted aryl is as defined herein.
[0068] "Arylthio" refers to the group --S-aryl, where aryl is as
defined herein.
[0069] "Substituted arylthio" refers to the group --S-(substituted
aryl), where substituted aryl is as defined herein.
[0070] "Carbonyl" refers to the divalent group --C(O)-- which is
equivalent to --C(.dbd.O)--.
[0071] "Carboxyl" or "carboxy" refers to --COOH or salts
thereof.
[0072] "Carboxyl ester" or "carboxy ester" refers to the groups
--C(O)O-alkyl, --C(O)O-substituted alkyl, --C(O)O-alkenyl,
--C(O)O-substituted alkenyl, --C(O)O-alkynyl, --C(O)O-substituted
alkynyl, --C(O)O-aryl, --C(O)O-substituted aryl,
--C(O)O-cycloalkyl, --C(O)O-substituted cycloalkyl,
--C(O)O-cycloalkenyl, --C(O)O-substituted cycloalkenyl,
--C(O)O-heteroaryl, --C(O)O-substituted heteroaryl,
--C(O)O-heterocyclic, and --C(O)O-substituted heterocyclic wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0073] "(Carboxyl ester)amino" refers to the group
--NR.sup.47C(O)O-alkyl, --NR.sup.47C(O)O-substituted alkyl,
--NR.sup.47C(O)O-alkenyl, --NR.sup.47C(O)O-substituted alkenyl,
--NR.sup.47C(O)O-alkynyl, --NR.sup.47C(O)O-substituted alkynyl,
--NR.sup.47C(O)O-aryl, --NR.sup.47C(O)O-substituted aryl,
--NR.sup.47C(O)O-cycloalkyl, --NR.sup.47C(O)O-substituted
cycloalkyl, --NR.sup.47C(O)O-cycloalkenyl,
--NR.sup.47C(O)O-substituted cycloalkenyl,
--NR.sup.47C(O)O-heteroaryl, --NR.sup.47C(O)O-substituted
heteroaryl, --NR.sup.47C(O)O-heterocyclic, and
--NR.sup.47C(O)O-substituted heterocyclic wherein R.sup.47 is alkyl
or hydrogen, and wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0074] "(Carboxyl ester)oxy" refers to the group --O--C(O)O-alkyl,
--O--C(O)O-substituted alkyl, --O--C(O)O-alkenyl,
--O--C(O)O-substituted alkenyl, --O--C(O)O-alkynyl,
--O--C(O)O-substituted alkynyl, --O--C(O)O-aryl,
--O--C(O)O-substituted aryl, --O--C(O)O-cycloalkyl,
--O--C(O)O-substituted cycloalkyl, --O--C(O)O-cycloalkenyl,
--O--C(O)O-substituted cycloalkenyl, --O--C(O)O-heteroaryl,
--O--C(O)O-substituted heteroaryl, --O--C(O)O-heterocyclic, and
--O--C(O)O-substituted heterocyclic wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0075] A "composition" as used herein, intends an active agent,
such as a compound as disclosed herein and a carrier, inert or
active. The carrier can be, without limitation, solid such as a
bead or resin, or liquid, such as phosphate buffered saline.
[0076] Administration or treatment in "combination" refers to
administering two agents such that their pharmacological effects
are manifest at the same time. Combination does not require
administration at the same time or substantially the same time,
although combination can include such administrations.
[0077] "Cyano" refers to the group --CN.
[0078] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10
carbon atoms having single or multiple cyclic rings including
fused, bridged, and spiro ring systems. The fused ring can be an
aryl ring provided that the non aryl part is joined to the rest of
the molecule. Examples of suitable cycloalkyl groups include, for
instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and
cyclooctyl.
[0079] "Cycloalkenyl" refers to non-aromatic cyclic alkyl groups of
from 3 to 10 carbon atoms having single or multiple cyclic rings
and having at least one >C.dbd.C< ring unsaturation and
preferably from 1 to 2 sites of >C.dbd.C< ring
unsaturation.
[0080] "Substituted cycloalkyl" and "substituted cycloalkenyl"
refers to a cycloalkyl or cycloalkenyl group having from 1 to 5 or
preferably 1 to 3 substituents selected from the group consisting
of oxo, thioxo, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
substituted sulfonyloxy, thioacyl, thiol, alkylthio, and
substituted alkylthio, wherein said substituents are as defined
herein.
[0081] "Cycloalkyloxy" refers to --O-cycloalkyl.
[0082] "Substituted cycloalkyloxy refers to --O-(substituted
cycloalkyl).
[0083] "Cycloalkylthio" refers to --S-cycloalkyl.
[0084] "Substituted cycloalkylthio" refers to --S-(substituted
cycloalkyl).
[0085] "Cycloalkenyloxy" refers to --O-cycloalkenyl.
[0086] "Substituted cycloalkenyloxy" refers to --O-(substituted
cycloalkenyl).
[0087] "Cycloalkenylthio" refers to --S-cycloalkenyl.
[0088] "Substituted cycloalkenylthio" refers to --S-(substituted
cycloalkenyl).
[0089] "Guanidino" refers to the group --NHC(.dbd.NH)NH.sub.2.
[0090] "Substituted guanidino" refers to
--NR.sup.53C(.dbd.NR.sup.53)N(R.sup.53).sub.2 where each R.sup.53
is independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, cycloalkyl, substituted cycloalkyl,
heterocyclic, and substituted heterocyclic and two R.sup.53 groups
attached to a common guanidino nitrogen atom are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, provided that at least one R.sup.53
is not hydrogen, and wherein said substituents are as defined
herein.
[0091] "Halo" or "halogen" refers to fluoro, chloro, bromo and
iodo.
[0092] "Hydroxy" or "hydroxyl" refers to the group --OH.
[0093] "Heteroaryl" refers to an aromatic group of from 1 to 10
carbon atoms and 1 to 4 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur within the ring. Such
heteroaryl groups can have a single ring (e.g., pyridinyl or furyl)
or multiple condensed rings (e.g., indolizinyl or benzothienyl)
wherein the condensed rings may or may not be aromatic and/or
contain a heteroatom provided that the point of attachment is
through an atom of the aromatic heteroaryl group. In one
embodiment, the nitrogen and/or the sulfur ring atom(s) of the
heteroaryl group are optionally oxidized to provide for the N-oxide
(N.fwdarw.O), sulfinyl, or sulfonyl moieties. Certain non-limiting
examples include pyridinyl, pyrrolyl, indolyl, thiophenyl,
oxazolyl, thizolyl, and furanyl.
[0094] "Substituted heteroaryl" refers to heteroaryl groups that
are substituted with from 1 to 5, preferably 1 to 3, or more
preferably 1 to 2 substituents selected from the group consisting
of the same group of substituents defined for substituted aryl.
[0095] "Heteroaryloxy" refers to --O-heteroaryl.
[0096] "Substituted heteroaryloxy" refers to the group
--O-(substituted heteroaryl).
[0097] "Heteroarylthio" refers to the group --S-heteroaryl.
[0098] "Substituted heteroarylthio" refers to the group
--S-(substituted heteroaryl).
[0099] "Heterocycle" or "heterocyclic" or "heterocycloalkyl" or
"heterocyclyl" refers to a saturated or partially saturated, but
not aromatic, group having from 1 to 10 ring carbon atoms and from
1 to 4 ring heteroatoms selected from the group consisting of
nitrogen, sulfur, or oxygen. Heterocycle encompasses single ring or
multiple condensed rings, including fused bridged and spiro ring
systems. In fused ring systems, one or more the rings can be
cycloalkyl, aryl, or heteroaryl provided that the point of
attachment is through a non-aromatic ring. In one embodiment, the
nitrogen and/or sulfur atom(s) of the heterocyclic group are
optionally oxidized to provide for the N-oxide, sulfinyl, or
sulfonyl moieties.
[0100] "Substituted heterocyclic" or "substituted heterocycloalkyl"
or "substituted heterocyclyl" refers to heterocyclyl groups that
are substituted with from 1 to 5 or preferably 1 to 3 of the same
substituents as defined for substituted cycloalkyl.
[0101] "Heterocyclyloxy" refers to the group --O-heterocycyl.
[0102] "Substituted heterocyclyloxy" refers to the group
--O-(substituted heterocycyl).
[0103] "Heterocyclylthio" refers to the group --S-heterocycyl.
[0104] "Substituted heterocyclylthio" refers to the group
--S-(substituted heterocycyl).
[0105] Examples of heterocycle and heteroaryls include, but are not
limited to, azetidine, pyrrole, furan, thiophene, imidazole,
pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,
isoindole, indole, dihydroindole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthylpyridine,
quinoxaline, quinazoline, cinnoline, pteridine, carbazole,
carboline, phenanthridine, acridine, phenanthroline, isothiazole,
phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine,
imidazoline, piperidine, piperazine, indoline, phthalimide,
1,2,3,4-tetrahydroisoquinoline,
4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine,
thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also
referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl,
piperidinyl, pyrrolidine, and tetrahydrofuranyl.
[0106] "Nitro" refers to the group --NO.sub.2.
[0107] "Oxo" refers to the atom (.dbd.O).
[0108] Phenylene refers to a divalent aryl ring, where the ring
contains 6 carbon atoms.
[0109] Substituted phenylene refers to phenylenes which are
substituted with 1 to 4, preferably 1 to 3, or more preferably 1 to
2 substituents selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
substituted sulfonyloxy, thioacyl, thiol, alkylthio, and
substituted alkylthio, wherein said substituents are as defined
herein.
[0110] "Spirocycloalkyl" and "spiro ring systems" refers to
divalent cyclic groups from 3 to 10 carbon atoms having a
cycloalkyl or heterocycloalkyl ring with a spiro union (the union
formed by a single atom which is the only common member of the
rings) as exemplified by the following structure:
##STR00005##
[0111] "Sulfonyl" refers to the divalent group --S(O).sub.2--.
[0112] "Substituted sulfonyl" refers to the group --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-alkenyl,
--SO.sub.2-substituted alkenyl, --SO.sub.2-cycloalkyl,
--SO.sub.2-substituted cycloalkyl, --SO.sub.2-cycloalkenyl,
--SO.sub.2-substituted cylcoalkenyl, --SO.sub.2-aryl,
--SO.sub.2-substituted aryl, --SO.sub.2-heteroaryl,
--SO.sub.2-substituted heteroaryl, --SO.sub.2-heterocyclic,
--SO.sub.2-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein. Substituted sulfonyl includes groups such as
methyl-SO.sub.2--, phenyl-SO.sub.2--, and
4-methylphenyl-SO.sub.2--.
[0113] "Substituted sulfonyloxy" refers to the group
--OSO.sub.2-alkyl, --OSO.sub.2-substituted alkyl,
--OSO.sub.2-alkenyl, --OSO.sub.2-substituted alkenyl,
--OSO.sub.2-cycloalkyl, --OSO.sub.2-substituted cycloalkyl,
--OSO.sub.2-cycloalkenyl, --OSO.sub.2-substituted cylcoalkenyl,
--OSO.sub.2-aryl, --OSO.sub.2-substituted aryl,
--OSO.sub.2-heteroaryl, --OSO.sub.2-substituted heteroaryl,
--OSO.sub.2-heterocyclic, --OSO.sub.2-substituted heterocyclic,
wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0114] "Thioacyl" refers to the groups H--C(S)--, alkyl-C(S)--,
substituted alkyl-C(S)--, alkenyl-C(S)--, substituted
alkenyl-C(S)--, alkynyl-C(S)--, substituted alkynyl-C(S)--,
cycloalkyl-C(S)--, substituted cycloalkyl-C(S)--,
cycloalkenyl-C(S)--, substituted cycloalkenyl-C(S)--, aryl-C(S)--,
substituted aryl-C(S)--, heteroaryl-C(S)--, substituted
heteroaryl-C(S)--, heterocyclic-C(S)--, and substituted
heterocyclic-C(S)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0115] "Thiol" refers to the group --SH.
[0116] "Thiocarbonyl" refers to the divalent group --C(S)-- which
is equivalent to --C(.dbd.S)--.
[0117] "Thioxo" refers to the atom (.dbd.S).
[0118] "Alkylthio" refers to the group --S-alkyl wherein alkyl is
as defined herein.
[0119] "Substituted alkylthio" refers to the group --S-(substituted
alkyl) wherein substituted alkyl is as defined herein.
[0120] "Optionally substituted" refers to a group selected from
that group and a substituted form of that group. Substituted groups
are defined herein. In one embodiment, substituents are selected
from C.sub.1-C.sub.10 or C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.6-C.sub.10 aryl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.10 heterocyclyl,
C.sub.1-C.sub.10 heteroaryl, halo, nitro, cyano, --CO.sub.2H or a
C.sub.1-C.sub.6 alkyl ester thereof.
[0121] "Tautomer" refer to alternate forms of a compound that
differ in the position of a proton, such as enol-keto and
imine-enamine tautomers, or the tautomeric forms of heteroaryl
groups containing a ring atom attached to both a ring --NH-- moiety
and a ring .dbd.N-- moiety such as pyrazoles, imidazoles,
benzimidazoles, triazoles, and tetrazoles.
[0122] "Uracil isostere" refers to an isostere of uracil. Such a
moiety provides some or all of the hydrogen bond
acceptor-donor-acceptor property of uracil and optionally provides
other structural characteristics of uracil. A skilled artisan will
further appreciate the meaning of this term by reading the non
limiting examples of such uracil isosteres provided herein.
[0123] As used herein, the term stereochemically pure denotes a
compound which has 80% or greater by weight of the indicated
stereoisomer and 20% or less by weight of other stereoisomers. In a
further embodiment, the compound of formula (I), (II), or (III) has
90% or greater by weight of the stated stereoisomer and 10% or less
by weight of other stereoisomers. In a yet further embodiment, the
compound of formula (I) has 95% or greater by weight of the stated
stereoisomer and 5% or less by weight of other stereoisomers. In a
still further embodiment, the compound of formula (I), (II), or
(III) has 97% or greater by weight of the stated stereoisomer and
3% or less by weight of other stereoisomers.
[0124] "Pharmaceutically acceptable salt" refers to salts of a
compound, which salts are suitable for pharmaceutical use and are
derived from a variety of organic and inorganic counter ions well
known in the art and include, when the compound contains an acidic
functionality, by way of example only, sodium, potassium, calcium,
magnesium, ammonium, and tetraalkylammonium; and when the molecule
contains a basic functionality, salts of organic or inorganic
acids, such as hydrochloride, hydrobromide, tartrate, mesylate,
acetate, maleate, and oxalate (see Stahl and Wermuth, eds.,
"Handbook of Pharmaceutically Acceptable Salts," (2002), Verlag
Helvetica Chimica Acta, Zurich, Switzerland), for a discussion of
pharmaceutical salts, their selection, preparation, and use.
[0125] Generally, pharmaceutically acceptable salts are those salts
that retain substantially one or more of the desired
pharmacological activities of the parent compound and which are
suitable for in vivo administration. Pharmaceutically acceptable
salts include acid addition salts formed with inorganic acids or
organic acids. Inorganic acids suitable for forming
pharmaceutically acceptable acid addition salts include, by way of
example and not limitation, hydrohalide acids (e.g., hydrochloric
acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid,
nitric acid, phosphoric acid, and the like.
[0126] Organic acids suitable for forming pharmaceutically
acceptable acid addition salts include, by way of example and not
limitation, acetic acid, trifluoroacetic acid, propionic acid,
hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic
acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic
acid, maleic acid, fumaric acid, tartaric acid, citric acid,
palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid,
cinnamic acid, mandelic acid, alkylsulfonic acids (e.g.,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic
acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids
(e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid,
2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic
acid, etc.), glutamic acid, hydroxynaphthoic acid, salicylic acid,
stearic acid, muconic acid, and the like.
[0127] Pharmaceutically acceptable salts also include salts formed
when an acidic proton present in the parent compound is either
replaced by a metal ion (e.g., an alkali metal ion, an alkaline
earth metal ion, or an aluminum ion) or by an ammonium ion (e.g.,
an ammonium ion derived from an organic base, such as,
ethanolamine, diethanolamine, triethanolamine, morpholine,
piperidine, dimethylamine, diethylamine, triethylamine, and
ammonia).
[0128] An "effective amount" is an amount sufficient to effect
beneficial or desired results. An effective amount can be
administered in one or more administrations, applications or
dosages. Such delivery is dependent on a number of variables
including the time period for which the individual dosage unit is
to be used, the bioavailability of the therapeutic agent, the route
of administration, etc. It is understood, however, that specific
dose levels of the therapeutic agents disclosed herein for any
particular subject depends upon a variety of factors including the
activity of the specific compound employed, bioavailability of the
compound, the route of administration, the age of the animal and
its body weight, general health, sex, the diet of the animal, the
time of administration, the rate of excretion, the drug
combination, and the severity of the particular disorder being
treated and form of administration. In general, one will desire to
administer an amount of the compound that is effective to achieve a
serum level commensurate with the concentrations found to be
effective in vivo. These considerations, as well as effective
formulations and administration procedures are well known in the
art and are described in standard textbooks. Consistent with this
definition and as used herein, the term "therapeutically effective
amount" is an amount sufficient to treat a specified disorder or
disease or alternatively to obtain a pharmacological response such
as inhibiting dUTPase.
[0129] As used herein, "treating" or "treatment" of a disease in a
patient refers to (1) preventing the symptoms or disease from
occurring in an animal that is predisposed or does not yet display
symptoms of the disease; (2) inhibiting the disease or arresting
its development; or (3) ameliorating or causing regression of the
disease or the symptoms of the disease. As understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. For the purposes of this
technology, beneficial or desired results can include one or more,
but are not limited to, alleviation or amelioration of one or more
symptoms, diminishment of extent of a condition (including a
disease), stabilized (i.e., not worsening) state of a condition
(including disease), delay or slowing of condition (including
disease), progression, amelioration or palliation of the condition
(including disease), states and remission (whether partial or
total), whether detectable or undetectable.
[0130] "dUTPase" means any of the following, which are considered
to be synonymous, "deoxyuridine triphosphate nucleotidohydrolase",
"deoxyuridine triphosphate pyrophosphatase", "dUTP
nucleotidohydrolase", "dUTP pyrophosphatase", and other equivalent
nomenclature for the dUTPase enzyme. In one aspect, dUTPase intends
DUT-N and DUT-M. In other aspects, it is DUT-N only, or
alternatively, DUT-M only. The amino acid and coding sequences for
dUTPase are known in the art and disclosed in U.S. Pat. No.
5,962,246. Methods for expressing and screening for expression
level of the enzyme are disclosed in U.S. Pat. No. 5,962,246 and
Ladner et al. (US Patent Publ. No. 2011/0212467A1).
[0131] "DUT-N" means the nuclear form of dUTPase.
[0132] "DUT-M" means the mitochondrial or cytoplasmic form of
dUTPase.
[0133] "dUTPase-directed therapy" intends therapeutics that target
the dUTPase pathway, e.g., in the case of cancer, e.g. TS-directed
therapies and the fluoropyrimidines (such as 5-FU), pemetrexed
(Alimta.RTM.), capecitabine (Xeloda.RTM.), S-1 and antifolates
(such as methotrexate) and chemical equivalents thereof.
Non-limiting examples include 5-flurouracil (5-FU), TS-directed
therapies and 5-FU based adjuvant therapy. Combination therapies
can include any intervention that alters nucleotide pools and/or
sensitizes the immune cells or viruses to the dUTPase inhibitor, as
are well known to the skilled artisan. For rheumatoid arthritis,
for example, the combination can be with an dihydrofolate reductase
(DHFR) inhibitor such as methotrexate.
[0134] 5-fluorouracil (5-FU) belongs to the family of therapy drugs
called pyrimidine based anti-metabolites. It is a pyrimidine
analog, which is transformed into different cytotoxic metabolites
that are then incorporated into DNA and RNA thereby inducing cell
cycle arrest and apoptosis. Chemical equivalents are pyrimidine
analogs which result in disruption of DNA replication. Chemical
equivalents inhibit cell cycle progression at S phase resulting in
the disruption of cell cycle and consequently apoptosis.
Equivalents to 5-FU include prodrugs, analogs and derivative
thereof such as 5'-deoxy-5-fluorouridine (doxifluoroidine),
1-tetrahydrofuranyl-5-fluorouracil (ftorafur), capecitabine
(Xeloda.RTM.), S-1 (MBMS-247616, consisting of tegafur and two
modulators, a 5-chloro-2,4-dihydroxypyridine and potassium
oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337),
LY231514 and ZD9331, as described for example in Papamicheal (1999)
The Oncologist 4:478-487.
[0135] "5-FU based adjuvant therapy" refers to 5-FU alone or
alternatively the combination of 5-FU with other treatments, that
include, but are not limited to radiation, methyl-CCNU, leucovorin,
oxaliplatin, irinotecin, mitomycin, cytarabine, levamisole.
Specific treatment adjuvant regimens are known in the art as
FOLFOX, FOLFOX4, FOLFIR1, MOF (semustine (methyl-CCNU), vincrisine
(Oncovin.RTM.) and 5-FU). For a review of these therapies see
Beaven and Goldberg (2006) Oncology 20(5):461-470. An example of
such is an effective amount of 5-FU and Leucovorin. Other
chemotherapeutics can be added, e.g., oxaliplatin or
irinotecan.
[0136] Capecitabine is a prodrug of (5-FU) that is converted to its
active form by the tumor-specific enzyme PynPase following a
pathway of three enzymatic steps and two intermediary metabolites,
5'-deoxy-5-fluorocytidine (5'-DFCR) and 5'-deoxy-5-fluorouridine
(5'-DFUR). Capecitabine is marketed by Roche under the trade name
Xeloda.RTM..
[0137] Leucovorin (Folinic acid) is an adjuvant used in cancer
therapy. It is used in synergistic combination with 5-FU to improve
efficacy of the chemotherapeutic agent. Without being bound by
theory, addition of Leucovorin is believed to enhance efficacy of
5-FU by inhibiting thymidylate synthase. It has been used as an
antidote to protect normal cells from high doses of the anticancer
drug methotrexate and to increase the antitumor effects of
fluorouracil (5-FU) and tegafur-uracil. It is also known as
citrovorum factor and Wellcovorin. This compound has the chemical
designation of L-Glutamic acid
N[4[[(2-amino-5-formyl1,4,5,6,7,8hexahydro4oxo6-pteridinyl)methyl]amino]b-
-enzoyl], calcium salt (1:1).
[0138] "Oxaliplatin" (Eloxatin) is a platinum-based chemotherapy
drug in the same family as cisplatin and carboplatin. It is
typically administered in combination with fluorouracil and
leucovorin in a combination known as FOLFOX for the treatment of
colorectal cancer. Compared to cisplatin, the two amine groups are
replaced by cyclohexyldiamine for improved antitumour activity. The
chlorine ligands are replaced by the oxalato bidentate derived from
oxalic acid in order to improve water solubility. Equivalents to
Oxaliplatin are known in the art and include, but are not limited
to cisplatin, carboplatin, aroplatin, lobaplatin, nedaplatin, and
JM-216 (see McKeage et al. (1997) J. Clin. Oncol. 201:1232-1237 and
in general, Chemotherapy for Gynecological Neoplasm, Curr. Therapy
and Novel Approaches, in the Series Basic and Clinical Oncology,
Angioli et al. Eds., 2004).
[0139] "FOLFOX" is an abbreviation for a type of combination
therapy that is used to treat cancer. This therapy includes 5-FU,
oxaliplatin and leucovorin. "FOLFIRI" is an abbreviation for a type
of combination therapy that is used treat cancer and comprises, or
alternatively consists essentially of, or yet further consists of
5-FU, leucovorin, and irinotecan. Information regarding these
treatments are available on the National Cancer Institute's web
site, cancer.gov, last accessed on Jan. 16, 2008.
[0140] Irinotecan (CPT-11) is sold under the trade name of
Camptosar. It is a semi-synthetic analogue of the alkaloid
camptothecin, which is activated by hydrolysis to SN-38 and targets
topoisomerase I. Chemical equivalents are those that inhibit the
interaction of topoisomerase I and DNA to form a catalytically
active topoisomerase I-DNA complex. Chemical equivalents inhibit
cell cycle progression at G2-M phase resulting in the disruption of
cell proliferation.
[0141] The term "adjuvant" therapy refers to administration of a
therapy or chemotherapeutic regimen to a patient after removal of a
tumor by surgery. Adjuvant therapy is typically given to minimize
or prevent a possible cancer reoccurrence. Alternatively,
"neoadjuvant" therapy refers to administration of therapy or
chemotherapeutic regimen before surgery, typically in an attempt to
shrink the tumor prior to a surgical procedure to minimize the
extent of tissue removed during the procedure.
[0142] The phrase "first line" or "second line" or "third line"
etc., refers to the order of treatment received by a patient. First
line therapy regimens are treatments given first, whereas second or
third line therapy are given after the first line therapy or after
the second line therapy, respectively. The National Cancer
Institute defines first line therapy as "the first treatment for a
disease or condition. In patients with cancer, primary treatment
can be surgery, chemotherapy, radiation therapy, or a combination
of these therapies. First line therapy is also referred to those
skilled in the art as primary therapy and primary treatment." See
National Cancer Institute website as www.cancer.gov, last visited
on May 1, 2008. Typically, a patient is given a subsequent
chemotherapy regimen because the patient did not shown a positive
clinical or sub-clinical response to the first line therapy or the
first line therapy has stopped.
[0143] As used herein, the term "antifolate" intends a drug or
biologic that impairs the function of folic acids, e.g., an
antimetabolite agent that inhibits the use of a metabolite, i.e.
another chemical that is part of normal metabolism. In cancer
treatment, antimetabolites interfere with DNA production, thus cell
division and growth of the tumor. Non-limiting examples of these
agents are dihydrofolate reductase inhibitors, such as
methotrexate, Aminopterin, and Pemetrexed; thymidylate synthase
inhibitors, such as Raltitrexed or Pemetrexed; purine based, i.e.
an adenosine deaminase inhibitor, such as Pentostatin, a
thiopurine, such as Thioguanine and Mercaptopurine, a
halogenated/ribonucleotide reductase inhibitor, such as Cladribine,
Clofarabine, Fludarabine, or a guanine/guanosine: thiopurine, such
as Thioguanine; or Pyrimidine based, i.e. cytosine/cytidine:
hypomethylating agent, such as Azacitidine and Decitabine, a DNA
polymerase inhibitor, such as Cytarabine, a ribonucleotide
reductase inhibitor, such as Gemcitabine, or a thymine/thymidine:
thymidylate synthase inhibitor, such as a Fluorouracil (5-FU).
[0144] In one aspect, the term "chemical equivalent" means the
ability of the chemical to selectively interact with its target
protein, DNA, RNA or fragment thereof as measured by the
inactivation of the target protein, incorporation of the chemical
into the DNA or RNA or other suitable methods. Chemical equivalents
include, but are not limited to, those agents with the same or
similar biological activity and include, without limitation a
pharmaceutically acceptable salt or mixtures thereof that interact
with and/or inactivate the same target protein, DNA, or RNA as the
reference chemical.
[0145] The terms "oligonucleotide" or "polynucleotide" or
"portion," or "segment" thereof refer to a stretch of
polynucleotide residues which is long enough to use in PCR or
various hybridization procedures to identify or amplify identical
or related parts of mRNA or DNA molecules. The polynucleotide
compositions of this invention include RNA, cDNA, genomic DNA,
synthetic forms, and mixed polymers, both sense and antisense
strands, and may be chemically or biochemically modified or may
contain non-natural or derivatized nucleotide bases, as will be
readily appreciated by those skilled in the art. Such modifications
include, for example, labels, methylation, substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,
etc.), charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),
intercalators (e.g., acridine, psoralen, etc.), chelators,
alkylators, and modified linkages (e.g., alpha anomeric nucleic
acids, etc.). Also included are synthetic molecules that mimic
polynucleotides in their ability to bind to a designated sequence
via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in
which peptide linkages substitute for phosphate linkages in the
backbone of the molecule.
[0146] When a genetic marker, e.g., over expression of dUTPase, is
used as a basis for selecting a patient for a treatment described
herein, the genetic marker is measured before and/or during
treatment, and the values obtained are used by a clinician in
assessing any of the following: (a) probable or likely suitability
of an individual to initially receive treatment(s); (b) probable or
likely unsuitability of an individual to initially receive
treatment(s); (c) responsiveness to treatment; (d) probable or
likely suitability of an individual to continue to receive
treatment(s); (e) probable or likely unsuitability of an individual
to continue to receive treatment(s); (f) adjusting dosage; (g)
predicting likelihood of clinical benefits; or (h) toxicity. As
would be well understood by one in the art, measurement of the
genetic marker in a clinical setting is a clear indication that
this parameter was used as a basis for initiating, continuing,
adjusting and/or ceasing administration of the treatments described
herein.
[0147] "Cancer" is a known medically as a malignant neoplasm, is a
broad group of diseases involving unregulated cell growth. In
cancer, cells divide and grow uncontrollably, forming malignant
tumors, and invade nearby parts of the body. Non-limiting examples
include colon cancer, colorectal cancer, gastric cancer, esophogeal
cancer, head and neck cancer, breast cancer, lung cancer, stomach
cancer, liver cancer, gall bladder cancer, or pancreatic cancer or
leukemia.
Compounds
[0148] In one aspect, provided herein are compounds of formula (I),
(II), and (III):
##STR00006##
or a tautomer thereof, or a pharmaceutically acceptable salt of
each thereof, wherein
##STR00007##
is a uracil isostere or a halo uracil;
##STR00008##
is uracil, halo uracil, or a uracil isostere; W is a bond or
optionally substituted --CH.sub.2--; W.sup.1 is a bond, N, or an
optionally substituted CH group; X is a bond, O, S, NR.sup.19,
optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.2-C.sub.6 alkenylene, or optionally substituted
C.sub.2-C.sub.6 alkynylene group, a divalent optionally substituted
C.sub.6-C.sub.10 aromatic hydrocarbon group, or a divalent
optionally substituted saturated or unsaturated C.sub.2-C.sub.10
heterocyclic or optionally substituted C.sub.1-C.sub.10 heteroaryl
group; R.sup.19 is hydrogen, optionally substituted C.sub.1-C.sub.6
alkyl or optionally substituted C.sub.3-C.sub.8 cycloalkyl; Y is a
bond or an optionally substituted C.sub.1-C.sub.10 alkylene which
further optionally has a cycloalkylidene structure on one carbon
atom, or is optionally substituted C.sub.2-C.sub.6 alkenylene, or
optionally substituted C.sub.2-C.sub.6 alkynylene group, or Y is
--L.sup.10-B.sup.1-L.sup.11-; L.sup.10 and L.sup.11 in dependently
are optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.2-C.sub.6 alkenylene, or optionally substituted
C.sub.2-C.sub.6 alkynylene group; B.sup.1 is a divalent optionally
substituted C.sub.6-C.sub.10 aromatic hydrocarbon group, or a
divalent optionally substituted saturated or unsaturated
C.sub.2-C.sub.10 heterocyclic or optionally substituted
C.sub.1-C.sub.10 heteroaryl group; Z is
--PO.sub.2--NR.sup.31R.sup.32, --SO.sub.2NR.sup.31R.sup.32,
--NR.sup.3PO.sub.2--R.sup.4, --NR.sup.3SO.sub.2--R.sup.4, or
R.sup.4 wherein R.sup.31 and R.sup.32 are the same or different and
each represents a hydrogen atom, optionally substituted
C.sub.1-C.sub.6 alkyl group optionally substituted with an aryl
group, wherein the aryl group, together with the R.sup.31 or
R.sup.32, may form a condensed bicyclic hydrocarbon, or R.sup.31
and R.sup.32 are taken together with the adjacent nitrogen atom
form an optionally substituted C.sub.2-C.sub.10 heterocyclic group
or an optionally substituted C.sub.1-C.sub.10 heteroaryl group;
Z.sup.1 is --PO.sub.2--NR.sup.31R.sup.32 or
--(OR.sup.3)P(O)--R.sup.4 wherein R.sup.31 and R.sup.32 are
independently a hydrogen atom, optionally substituted
C.sub.1-C.sub.6 alkyl group optionally substituted with an aryl
group, wherein the aryl group, together with the R.sup.31 or
R.sup.32, may form a condensed bicyclic hydrocarbon, or R.sup.31
and R.sup.32 taken together with the adjacent nitrogen atom form an
optionally substituted C.sub.2-C.sub.10 heterocyclic group or an
optionally substituted C.sub.1-C.sub.10 heteroaryl group; R.sup.3
is hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl; and
R.sup.4 is optionally substituted C.sub.6-C.sub.10 aryl, an
optionally substituted C.sub.2-C.sub.10 heterocyclic group, or an
optionally substituted C.sub.1-C.sub.10 heteroaryl group.
[0149] In one embodiment, provided herein is a compound of formula
(III):
##STR00009##
wherein A is an uracil isostere selected from:
##STR00010##
R.sup.10 is hydrogen, R.sup.12, or --O--R.sup.12, R.sup.12 is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl optionally substituted with 1-3 hydroxy, fluoro, chloro,
and amino substituent, R.sup.11 is hydrogen, halo, R.sup.12 or
--O--R.sup.12, wherein R.sup.12 is defined as above, r is 1, 2, or
3,
L.sup.1- is
##STR00011##
[0150] wherein
Y.sup.1 is CH.sub.2, O, S,
X.sup.10 is NH, NCO.sub.2R.sup.20, O, or CH.sub.2,
[0151] R.sup.20 is C.sub.1-C.sub.6 alkyl optionally substituted
with 1-3 C.sub.6-C.sub.10 aryl groups, u is 0, 1, 2, 3, or 4,
R.sup.z is hydroxy or hydrogen, R.sup.w is C.sub.1-C.sub.6 alkyl or
hydrogen, and the phenylene and the heteroarylene rings are
optionally substituted, Z is phenyl or a 5 or 6 member heteroaryl
substituted with an R.sup.6 and an R.sup.60 groups, wherein the
R.sup.6 and the R.sup.60 are positioned 1,2 with respect to each
other, R.sup.6 is hydrogen, optionally substituted C.sub.1-C.sub.6
alkoxy, or halo, and
R.sup.60 is --OR.sup.7 or --NHR.sup.7R.sup.70,
[0152] R.sup.7 is optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted C.sub.3-C.sub.10
heteroaryl, optionally substituted C.sub.3-C.sub.10 heterocyclyl,
or optionally substituted phenyl, and R.sup.70 is hydrogen or
R.sup.7.
[0153] In another embodiment, the uracil isostere is an optionally
substituted cycloalkyl or optionally substituted heterocyclyl ring
which is monocyclic, bicyclic, tricyclic, or tetracyclic, wherein
the ring comprises a moiety selected from
--C(.dbd.V)--NH--C(.dbd.V)--,
--C(.dbd.V)--CH.sub.2--C(.dbd.V)--.
[0154] In another embodiment, the uracil isostere is optionally
substituted meta-dihaho phenyl or optionally substituted
1,3-dihalosubstituted C.sub.3-C.sub.10 heteroaryl. In another
embodiment, the uracil isostere is optionally substituted
meta-difluoro phenyl or meta-fluoro-halo phenyl.
[0155] In certain embodiments, the uracil isotere is halo uracil.
In certain embodiments, the uracil isotere, particularly for
formulas (I) and (II) are not halo uracil. As used herein, halo
uracil refers to a halogenated uracil, a non limiting example of
which includes 5-halo uracil.
[0156] In another embodiment, the uracil isostere is of
formula:
##STR00012##
wherein each V independently is O or S, each R.sup.1 independently
is hydrogen, C.sub.1-C.sub.6 alkyl optionally substituted with
C.sub.3-C.sub.5 cycloalkyl, or C.sub.3-C.sub.5 cycloalkyl, each
R.sup.2 is independently --OH, --SH, --OR.sup.1, --SR.sup.1, or
halo wherein R.sup.1 is defined as above, each Q.sup.1 and Q.sup.2
independently are --CH.sub.2--, O, S or an oxidized form thereof,
NH or an oxidized form thereof, or Q.sup.1 and Q.sup.2 together
form a --CH.dbd.CH-- moiety; provided that Q.sup.1 and Q.sup.2 are
both not O, S or an oxidized form thereof, NH or an oxidized form
thereof or a combination of each thereof; wherein each --CH.dbd.,
--CH.sub.2--, and --NH-- is optionally substituted.
[0157] In another embodiment, R.sup.1 independently is hydrogen or
methyl. In another embodiment, each R.sup.1 is hydrogen. In another
embodiment, each R.sup.1 is methyl. In another embodiment, each V
independently is O. In another embodiment, each V independently is
S.
[0158] In another embodiment, each Q.sup.1 independently is O. In
another embodiment, each Q.sup.1 independently is S. In another
embodiment, each Q.sup.1 independently is optionally substituted
--CH.sub.2--. In another embodiment, each Q.sup.1 independently is
optionally substituted --NH--.
[0159] In another embodiment, each Q.sup.2 independently is O. In
another embodiment, each Q.sup.2 independently is S. In another
embodiment, each Q.sup.2 independently is optionally substituted
--CH.sub.2--. In another embodiment, each Q.sup.1 independently is
optionally substituted --NH--.
[0160] In another embodiment, the uracil isostere is:
##STR00013##
[0161] In some embodiments, the uracil isostere is:
##STR00014##
[0162] In some embodiments, the uracil isostere is:
##STR00015##
[0163] In some embodiments, the uracil isostere is:
##STR00016##
[0164] In some embodiments, the uracil isostere is:
##STR00017##
[0165] In another embodiment, --W--X--Y-- is
--CH.sub.2--X--SO.sub.2--NH--CH(R.sup.Y)--;
--CH.sub.2--X--SO.sub.2--NH--C(R.sup.Y).sub.2--; or
--CH.sub.2--X--B--CH.sub.2CR.sup.ZR.sup.W--,
X is optionally substituted C.sub.1-C.sub.6 alkylene wherein one of
the methylene groups within the alkylene chain is optionally
replaced with an O or S atom, such that X is optionally substituted
alkylene or optionally substituted heteroalkylene; B is a
optionally substituted C.sub.3-C.sub.10 heteroaryl; R.sup.Y an
R.sup.w are independently hydrogen or C.sub.1-C.sub.6 alkyl; and
R.sup.z is hydrogen or hydroxy.
[0166] In one embodiment, B is a 5 membered heteroaryl containing
up to 3 or 4 heteroatoms selected from nitrogen, sulfur and oxygen.
In one embodiment, B is:
##STR00018##
[0167] In another embodiment, --W--X--Y-- or L.sup.1 is
##STR00019##
wherein
X.sup.10 is NH, NCO.sub.2R.sup.20, O, or CH.sub.2,
[0168] R.sup.20 is C.sub.1-C.sub.6 alkyl optionally substituted
with 1-3 C.sub.6-C.sub.10 aryl groups, u is 0, 1, 2, 3, or 4,
Y.sup.1 is CH.sub.2, O or S,
[0169] R.sup.z is hydroxy or hydrogen, R.sup.w is C.sub.1-C.sub.6
alkyl or hydrogen, the phenylene and the heteroarylene rings are
optionally substituted.
[0170] In some embodiments, --W--X--Y-- or L.sup.1 is
##STR00020##
[0171] In some embodiments, --W--X--Y-- or L.sup.1 is
##STR00021##
[0172] In another embodiment, R.sup.4 is optionally substituted
C.sub.6-C.sub.10 aryl. In another embodiment, R.sup.4 is optionally
substituted C.sub.2-C.sub.10 heterocyclic group. In another
embodiment, R.sup.4 is optionally substituted C.sub.1-C.sub.10
heteroaryl group. In another embodiment, when Y is
-L.sup.10-B.sup.1-L.sup.11-, Z is R.sup.4.
[0173] In some embodiments, Z is phenyl or a 5 or 6 membered
heteroaryl substituted with an R.sup.6 and an R.sup.60 groups,
wherein the R.sup.6 and the R.sup.60 are positioned 1,2 with
respect to each other,
R.sup.6 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkoxy,
or halo, and
R.sup.60 is --OR.sup.7 or --NHR.sup.7R.sup.70,
[0174] R.sup.7 is optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.3-C.sub.5 cycloalkyl, optionally substituted C.sub.3-C.sub.10
heteroaryl, optionally substituted C.sub.3-C.sub.10 heterocyclyl,
or optionally substituted phenyl, and R.sup.70 is hydrogen or
R.sup.7.
[0175] In some embodiments, Z or R.sup.4 is selected from:
##STR00022##
wherein each R.sup.6 and R.sup.7 independently are defined as in
any aspect or embodiment above, each R.sup.61and R.sup.62
independently is N or CH, provided that at least one of R.sup.61and
R.sup.62 is N, each R.sup.63 independently is NR.sup.70, S, O, and
each R.sup.64 independently is N or CH.
[0176] In some embodiments, provided herein is a compound of
formula:
##STR00023##
wherein L.sub.1 is as defined above.
[0177] In some embodiments, provided herein is a compound of
formula:
##STR00024##
wherein L.sub.1 is as defined above.
[0178] In another embodiment, Z is:
##STR00025##
R.sup.6 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkoxy,
or halo, and R.sup.7 is optionally substituted C.sub.1-C.sub.6
alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.3-C.sub.5 cycloalkyl, optionally substituted
C.sub.3-C.sub.10heteroaryl, optionally substituted C.sub.3-C.sub.10
heterocyclyl, or optionally substituted phenyl.
[0179] In one embodiment, R.sup.6 is hydrogen. In one embodiment,
R.sup.6 is halo. In another embodiment, R.sup.6 is fluoro. In one
embodiment, R.sup.6 is C.sub.1-C.sub.6 alkoxy. In one embodiment,
R.sup.6 is C.sub.1-C.sub.6 alkoxy substituted with 1-3 fluoro
groups. In some embodiments, R.sup.6 is hydrogen, F, Cl, OMe, or
OCF.sub.3.
[0180] In one embodiment, R.sup.7 is C.sub.1-C.sub.6 alkyl
substituted with a C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.10
heterocyclyl, or C.sub.1-C.sub.10 heteroaryl. In one embodiment,
R.sup.7 is
##STR00026##
[0181] In one embodiment, R.sub.7 is C.sub.1-C.sub.6 alkyl
optionally substituted with a C.sub.3-C.sub.8 cycloalkyl, 4-8
membered heterocyclyl, or R.sup.7 is C.sub.1-C.sub.6 alkyl
substitute with 1-3 fluoro atoms.
[0182] In another embodiment, R.sup.7 is:
##STR00027##
wherein t is 1, 2, or 3. In another embodiment, t is 1. In another
embodiment, t is 2. In another embodiment, t is 3.
[0183] In another embodiment, the cycloalkyl is cyclopropyl. In
another embodiment, the cycloalkyl is cyclobutyl. In another
embodiment, the cycloalkyl is cyclopentyl. In another embodiment,
the cycloalkyl is cyclohexyl. In another embodiment, R.sup.7 is
isobutyl. In another embodiment, R.sup.7 is neopentyl.
[0184] In another embodiment, the heterocyclyl is
##STR00028##
[0185] In another embodiment, the heterocyclyl is:
##STR00029##
[0186] In another embodiment, the heterocyclyl is:
##STR00030##
[0187] In one embodiment, the compound is PCI10213 of formula:
##STR00031##
[0188] In another embodiment, the compound is of formula:
##STR00032##
wherein L.sub.1 is as defined above.
[0189] In another embodiment, the compound is of formula:
##STR00033##
wherein L.sub.1 is as defined above.
[0190] In another embodiment, the compound is of formula:
##STR00034##
wherein L.sub.1 is as defined above.
[0191] In another embodiment, the compound is of formula:
##STR00035##
wherein L.sub.1 is as defined above.
[0192] In one embodiment, provided herein is a compound of
formula:
##STR00036##
wherein A is selected from:
##STR00037##
X.sup.10 is NH, NCO.sub.2R.sup.20, O, or CH.sub.2;
[0193] R.sup.20 is C.sub.1-C.sub.6 alkyl optionally substituted
with 1-3 C.sub.6-C.sub.10 aryl groups; u is 0, 1, 2, 3, or 4;
R.sup.11 is hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, or C.sub.2-C.sub.6 alkynyl wherein each alkyl, alkenyl,
and alkynyl is optionally substituted with 1-3 hydroxy, fluoro,
chloro, and amino substituent; R.sub.60 is C.sub.1-C.sub.6 alkyl
and r is 1, 2, or 3.
[0194] In one embodiment, A is:
##STR00038##
[0195] In another embodiment, A is selected from:
##STR00039##
[0196] In another embodiment, X.sup.10 is CH.sub.2 or NH. In
another embodiment, t is 1. In another embodiment, t is 2. In
another embodiment, t is 3.
[0197] In another embodiment, provided herein is a compound
selected from:
##STR00040## ##STR00041##
and a diastereomer or an enantiomer thereof.
[0198] In another embodiment, provided here are the compounds:
##STR00042## ##STR00043## ##STR00044##
d pharmaceutically acceptable salts thereof.
[0199] The compounds provided herein include individual, separated
enantiomers and diastereomers, tautotomers, and pharmaceutically
acceptable salts of each thereof, wherever applicable. In one
aspect, the compounds are provided as stereochemical pure, e.g.,
PCI 10586 and pharmaceutically acceptable salts thereof, as
described herein. As used herein, the term stereochemically pure
denotes a compound which has 80% or greater by weight of the
indicated stereoisomer and 20% or less by weight of other
stereoisomers. In a further aspect, the compounds as described
herein have 90% or greater by weight of the denoted stereoisomer
and 10% or less by weight of other stereoisomers. In a yet further
embodiment, the compounds of this disclosure have 95% or greater by
weight of the denoted stereoisomer and 5% or less by weight of
other stereoisomers. In a still further embodiment, the compounds
have 97% or greater by weight of the denoted stereoisomer and 3% or
less by weight of other stereoisomers.
Synthesis
[0200] The following general synthetic scheme is used to prepare
the compounds provided herein. For example, compounds of formula I
are synthesized as shown in the reaction scheme below.
##STR00045##
In general, uracil, uracil isostere, or a halo uracil is treated
with a suitable base such as butyl lithium in a solvent such as
tetrahydrofuran or dimethylformamide. The A(-) anion can also be
generated by halogen exchange of an A-halo bond with an alkyl
lithium. It is then coupled with compound B, wherein LG is a
leaving group such as halogen, tosylate or mesylate to provide
compounds of formula (1). In some embodiments, protection of an NH,
OH, or such other group in uracil, uracil isostere, halo uracil, or
the --W--X--Y--Z moiety is required. Compounds of formula (III) can
also be synthesized in an analogous manner.
[0201] For example, compounds of formula II can be synthesized as
schematically illustrated below:
##STR00046##
[0202] Suitable conditions for the condensation reaction with the
keto group, dehydration, formation of the Wittig reagent and the
subsequent Witting reaction, and the Schiff's base formation are
well known to the skilled artisan.
##STR00047##
[0203] Illustrative and non-limiting synthesis of other compounds
containing other linkers, e.g., L1 or --W--X--Y--, are shown
above.
[0204] A-ring substituted compounds provided here are synthesized
as shown below and or following methods well known in the art in
view of the present disclosure. See also, Journal of Heterocyclic
Chemistry (2005) vol. 42, #2 p. 201-207, Journal of the American
Chemical Society (2009) vol. 131, p. 8196-8210, Journal of
Heterocyclic Chemistry (1994) vol. 31, #2 p. 565-568, and Journal
of Medicinal Chemistry (1994) vol. 37, #13 p. 2059-2070, each of
which is incorporated herein by reference.
##STR00048##
[0205] Additional --W--X--Y--Z moieties are disclosed in US
2011/0082163; US 2012/0225838; Miyahara et al., J. Med. Chem.
(2012) 55, 2970-2980; Miyakoshi et al., J. Med. Chem. (2012) 55,
2960-2969; Miyahara et al., J. Med. Chem. (2012) 55 (11), pp
5483-5496; and Miyakoshi et al., J. Med. Chem. (2012) 55 (14), pp
6427-6437 (each of which are incorporated herein by reference) and
can be used with the A moieties disclosed herein.
[0206] These and other compounds provided herein are synthesized
following art recognized methods with the appropriate substitution
of commercially available reagents as needed. For example, and
without limitation, methods for synthesizing certain other
compounds are described in US 2011/0082163; US 2012/0225838;
Miyahara et al., J. Med. Chem. (2012) 55, 2970-2980; Miyakoshi et
al., J. Med. Chem. (2012) 55, 2960-2969; Miyahara et al., J. Med.
Chem. (2012) 55 (11), pp 5483-5496; and Miyakoshi et al., J. Med.
Chem. (2012) 55 (14), pp 6427-6437 (each supra), which methods can
be adapted by the skilled artisan upon reading this disclosure
and/or based on synthetic methods well known in the art, to prepare
the compounds provided herein. Protection deprotection methods and
protecting groups useful for such purposes are well known in the
art, for example in Greene's Protective Groups in Organic
Synthesis, 4.sup.th Edition, Wiley, 2006, or a later edition of the
book.
[0207] The compounds and the intermediates are separated from the
reaction mixture, when desired, following art known methods such as
crystallization, chromatography, distillation, and the like. The
compounds and the intermediates are characterized by art known
methods such as thin layer chromatography, nuclear magnetic
resonance spectroscopy, high performance liquid chromatography, and
the like. As described in detail herein, a racemic mixture of the
compound can be separated to the diastereomers and tested and used
diagnostically or therapeutically as described herein. Thus, in one
aspect, the compound is provided as a stereochemically pure
enantiomer, e.g., PCI 10586 or PCI 10585, as described herein.
[0208] Methods of testing and using the compounds provided herein
are performed following art recognized in vitro (cell free), ex
vivo or in vivo methods. For example, and without limitation,
methods for testing and using certain other compounds are described
in US 2011/0082163; US 2012/0225838; Miyahara et al., J. Med. Chem.
(2012) 55, 2970-2980; Miyakoshi et al., J. Med. Chem. (2012) 55,
2960-2969; Miyahara et al., J. Med. Chem. (2012) 55 (11), pp
5483-5496; Miyakoshi et al., J. Med. Chem. (2012) 55 (14), pp
6427-6437 (each of which in incorporated by reference), which
methods can be adapted by the skilled artisan upon reading this
disclosure and/or based on methods well known in the art, to test
and use the compounds provided herein.
Compositions
[0209] Compositions, including pharmaceutical compositions
comprising the compounds described herein can be manufactured by
means of conventional mixing, dissolving, granulating,
dragee-making levigating, emulsifying, encapsulating, entrapping,
or lyophilization processes. The compositions can be formulated in
conventional manner using one or more physiologically acceptable
carriers, diluents, excipients, or auxiliaries which facilitate
processing of the compounds provided herein into preparations which
can be used pharmaceutically.
[0210] The compounds of the technology can be administered by
parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV,
intracisternal injection or infusion, subcutaneous injection, or
implant), oral, by inhalation spray nasal, vaginal, rectal,
sublingual, urethral (e.g., urethral suppository) or topical routes
of administration (e.g., gel, ointment, cream, aerosol, etc.) and
can be formulated, alone or together, in suitable dosage unit
formulations containing conventional non-toxic pharmaceutically
acceptable carriers, adjuvants, excipients, and vehicles
appropriate for each route of administration.
[0211] In one embodiment, this technology relates to a composition
comprising a compound as described herein and a carrier.
[0212] In another embodiment, this technology relates to a
pharmaceutical composition comprising a compound as described
herein and a pharmaceutically acceptable carrier.
[0213] In another embodiment, this technology relates to a
pharmaceutical composition comprising a therapeutically effective
amount of a compound as described herein and a pharmaceutically
acceptable carrier.
[0214] The pharmaceutical compositions for the administration of
the compounds can be conveniently presented in dosage unit form and
can be prepared by any of the methods well known in the art of
pharmacy. The pharmaceutical compositions can be, for example,
prepared by uniformly and intimately bringing the compounds
provided herein into association with a liquid carrier, a finely
divided solid carrier or both, and then, if necessary, shaping the
product into the desired formulation. In the pharmaceutical
composition the compound provided herein is included in an amount
sufficient to produce the desired therapeutic effect. For example,
pharmaceutical compositions of the technology may take a form
suitable for virtually any mode of administration, including, for
example, topical, ocular, oral, buccal, systemic, nasal, injection,
infusion, transdermal, rectal, and vaginal, or a form suitable for
administration by inhalation or insufflation.
[0215] For topical administration, the compounds can be formulated
as solutions, gels, ointments, creams, suspensions, etc., as is
well-known in the art.
[0216] Systemic formulations include those designed for
administration by injection (e.g., subcutaneous, intravenous,
infusion, intramuscular, intrathecal, or intraperitoneal injection)
as well as those designed for transdermal, transmucosal, oral, or
pulmonary administration.
[0217] Useful injectable preparations include sterile suspensions,
solutions, or emulsions of the compounds provided herein in aqueous
or oily vehicles. The compositions may also contain formulating
agents, such as suspending, stabilizing, and/or dispersing agents.
The formulations for injection can be presented in unit dosage
form, e.g., in ampules or in multidose containers, and may contain
added preservatives.
[0218] Alternatively, the injectable formulation can be provided in
powder form for reconstitution with a suitable vehicle, including
but not limited to sterile pyrogen free water, buffer, and dextrose
solution, before use. To this end, the compounds provided herein
can be dried by any art-known technique, such as lyophilization,
and reconstituted prior to use.
[0219] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are known in the art.
[0220] For oral administration, the pharmaceutical compositions may
take the form of, for example, lozenges, tablets, or capsules
prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (e.g., pregelatinised maize
starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose);
fillers (e.g., lactose, microcrystalline cellulose, or calcium
hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or
silica); disintegrants (e.g., potato starch or sodium starch
glycolate); or wetting agents (e.g., sodium lauryl sulfate). The
tablets can be coated by methods well known in the art with, for
example, sugars, films, or enteric coatings.
[0221] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions, and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents, and preserving agents in
order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the compounds provided herein in
admixture with non-toxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
can be for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents (e.g., corn starch or alginic
acid); binding agents (e.g. starch, gelatin, or acacia); and
lubricating agents (e.g., magnesium stearate, stearic acid, or
talc). The tablets can be left uncoated or they can be coated by
known techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate can be employed. They
may also be coated by the techniques well known to the skilled
artisan. The pharmaceutical compositions of the technology may also
be in the form of oil-in-water emulsions.
[0222] Liquid preparations for oral administration may take the
form of, for example, elixirs, solutions, syrups, or suspensions,
or they can be presented as a dry product for constitution with
water or other suitable vehicle before use. Such liquid
preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives, or hydrogenated
edible fats); emulsifying agents (e.g., lecithin, or acacia);
non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol,
Cremophore.TM., or fractionated vegetable oils); and preservatives
(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, preservatives,
flavoring, coloring, and sweetening agents as appropriate.
Use of Compounds for Preparing Medicaments
[0223] The compounds and compositions of the present invention are
also useful in the preparation of medicaments to treat a variety of
pathologies as described herein. The methods and techniques for
preparing medicaments of a composition are known in the art.
[0224] For the purpose of illustration only, pharmaceutical
formulations and routes of delivery are detailed herein.
[0225] Thus, one of skill in the art would readily appreciate that
any one or more of the compositions described above, including the
many specific embodiments, can be used by applying standard
pharmaceutical manufacturing procedures to prepare medicaments to
treat the many disorders described herein. Such medicaments can be
delivered to the subject by using delivery methods known in the
pharmaceutical arts.
Methods and Therapies
[0226] The compositions and compounds as disclosed herein are
useful in methods of inhibiting dUTPase or enhancing the efficacy
of a dUTPase-directed therapy, or yet further, reversing resistance
to dUTPase therapies. The methods comprise, or alternatively
consist essentially of, or yet further consist of, contacting the
dUTPase with an effective amount of the compound or composition as
disclosed herein. In one embodiment, the methods further comprise,
or alternatively consist essentially of, or yet further consist of,
contacting the dUTPase with an effective amount of a
dUTPase-directed therapy. In one aspect, the contacting of the
dUTPase-directed therapy is prior to, concurrent or subsequent to
contacting with the compound or composition of this disclosure.
[0227] One of skill in the art can also determine if the compound
or combination inhibits dUTPase in vitro by contacting the compound
or combination with purified or recombinant dUTPase in a cell free
system. The purified or recombinant dUTPase and can be from any
species, e.g., simian, canine, bovine, ovine, rat, mouse or human.
In one aspect, the dUTPase is DUT-N or DUT-M. Isolation,
characterization and expression of dUTPase isoforms are disclosed
in U.S. Pat. No. 5,962,246 and known in the art.
[0228] The contacting can be performed cell-free in vitro or ex
vivo with a cell or in a cell culture. When performed in vitro or
ex vivo, the compounds, compositions or agents can be directly
added to the enzyme solution or added to the cell culture medium.
When practiced in vitro or ex vivo, the method can be used to
screen for novel combination therapies, formulations or treatment
regimens, prior to administration to administration to an animal or
a human patient. Methods to quantify inhibition are known in the
art, see, U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467 and
U.S. Pat. No. 7,601,702. For example, a fixed dose of a dUTPase
directed therapy (e.g., 5-FU or Pemetrexed) can be added to the
system and varying amounts of the compound can be subsequently
added to system. Alternatively, a fixed dose of a compound of this
invention can be added to the system and varying amounts of the
dUTPase directed therapy (e.g., 5-FU or Pemetrexed) compound can be
subsequently added to system.
[0229] In one aspect, the contacting is ex vivo and the cell or
tissue to be contacted over expresses dUTPase. These cells can be
isolated from a patient prior to administration to the patient or
can be purchased from a depository such as the American Type
Culture Collection (ATCC). Non-limiting examples of animal (e.g.,
canine, an equine, a bovine, a feline, an ovine, a mouse, a rat or
a simian) and human cells that are known to over express dUTPase
include, without limitation cancer cells, e.g. colon cancer,
colorectal cancer, gastric cancer, head and neck cancer, breast
cancer, stomach cancer or lung cancer. The cancer can be metastatic
or non-metastatic. Methods to quantify inhibition are known in the
art, see, U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467 and
U.S. Pat. No. 7,601,702 and Wilson et al. (2012) Mol. Cancer Ther.
11:616-628.
[0230] When practiced in vivo in a patient such as an animal or
human, the compounds, compositions or agents are administered in an
effective amount by a suitable route of administration, as
determined by a treating physician taking into account the patient,
disease and other factors. When practiced in a non-human animal,
e.g., an appropriate mouse model, the method can be used to screen
for novel combination therapies, formulations or treatment
regimens, prior to administration to a human patient.
[0231] This disclosure also provides methods of treating a disease
whose treatment is impeded by the expression of dUTPase,
comprising, or alternatively consisting essentially of, or yet
further consisting of, administering to a patient in need of such
treatment an effective amount of the compound or composition of
this disclosure, thereby treating the disease. In one aspect, the
method further comprises isolating a cell or tissue sample from the
patient and screening for the expression level of dUTPase, wherein
over expression of dUTPase in the sample as compared to a control
sample serves as a basis for selecting the patient as suitable for
the method and therapies. Methods to quantify dUTPase are known in
the art. Effective amounts will vary with the patient, the disease
and the general health of the patient and are determined by the
treating physician. Methods to quantify inhibition are known in the
art, see, U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467 and
U.S. Pat. No. 7,601,702 and Wilson et al. (2012) Mol. Cancer Ther.
11:616-628. If the patient sample shows over expression of dUTPase,
the therapy is administered to the patient. If the patient sample
does not show over expression, an alternate therapy is chosen. The
screen can be repeated throughout therapy as a means to monitor the
therapy and/or dosage regimen.
[0232] To practice this method, the sample is a patient sample
containing the tumor tissue, normal tissue adjacent to said tumor,
normal tissue distal to said tumor or peripheral blood lymphocytes.
In a further aspect, the patient or patient population to be
treated also is treatment naive.
[0233] In one aspect, the method also requires isolating a sample
containing the genetic material to be tested; however, it is
conceivable that one of skill in the art will be able to analyze
and identify genetic markers in situ at some point in the future.
Accordingly, in one aspect, the inventions of this application are
not to be limited to requiring isolation of the genetic material
prior to analysis.
[0234] These methods also are not limited by the technique that is
used to identify the expression level or in aspects where
expression has been linked to a polymorphism, the polymorphism of
interest. Suitable methods include but are not limited to the use
of hybridization probes, antibodies, primers for PCR analysis, and
gene chips, slides and software for high throughput analysis.
Additional genetic markers can be assayed and used as negative
controls.
[0235] In one aspect, the subject or patient is an animal or a
human patient. Non-limiting examples of animals include a feline, a
canine, a bovine, an equine, an ovine, a mouse, a rat or a
simian.
[0236] Diseases in which treatment is impeded by the expression of
dUTPase include, without limitation, cancer, viral infection,
bacterial infection or an autoimmune disorder. For example, in
rheumatoid arthritis, inflammatory bowel disease or other
autoimmune disorders, a dUTPase inhibitor can be used in
combination with an antifolate or fluoropyrimidine or other
thymidylate synthase and dihydrofolate reductase inhibitors;
parasitic, viral or bacterial infections can be treated similarly
employing a combination therapy including a dUTPase inhibitor.
Non-limiting examples of cancer include, colon cancer, colorectal
cancer, gastric cancer, head and neck cancer, breast cancer,
stomach cancer, lung cancer or a leukemia. The cancer can be
metastatic or non-metastatic.
[0237] In one aspect, the compound or composition is administered
as one or more of: a first line therapy or alternatively, a second
line therapy, a third line therapy, or a fourth or subsequent line
therapy to administration of a dUPTase-directed therapy.
Non-limiting examples of dUTPase-directed therapies include an
antimetabolite or a fluoropyrmidine therapy or a 5-FU based
adjuvant therapy or an equivalent or each thereof, such as 5-FU,
tegafur, gimeracil, oteracil potassium, capcitabine,
5-fluoro-2'-deoxyuridine, methotrexate, or pemetrexed or an
equivalent of each thereof.
[0238] Certain compounds provided herein demonstrated substantial,
such as, 20-100% DUTPase inhibitory effect, e.g., an ability to
inhibit dUTPase under conditions described herein below, and/or
known to the skilled artisan, compared, for example, a compound
provided herein:
##STR00049##
In one embodiment, certain therapeutic methods provided herein
exclude the use of the compounds PCI 10898, 10897, 10928, and
10929.
Kits
[0239] The compounds and compositions, as described herein, can be
provided in kits. The kits can further contain additional dUTPase
inhibitors and optionally, instructions for use. In a further
aspect, the kit contains reagents and instructions to perform the
screen to identify patients more likely to respond to the therapy
as described above.
Screening Assays
[0240] This invention also provides screening assays to identify
potential therapeutic agents of known and new compounds and
combinations. For example, one of skill in the art can also
determine if the compound or combination inhibits dUTPase in vitro
by contacting the compound or combination with purified or
recombinant dUTPase in a cell free system. The purified or
recombinant dUTPase and can be from any species, e.g., simian,
canine, bovine, ovine, rat, mouse or human. In one aspect, the
dUTPase is DUT-N or DUT-M. Isolation, characterization and
expression of dUTPase isoforms are disclosed in U.S. Pat. No.
5,962,246 and known in the art.
[0241] The contacting can be performed cell-free in vitro or ex
vivo with a cell or in a cell culture. When performed in vitro or
ex vivo, the compounds, compositions or agents can be directly
added to the enzyme solution or added to the cell culture medium.
When practiced in vitro or ex vivo, the method can be used to
screen for novel combination therapies, formulations or treatment
regimens, prior to administration to administration to an animal or
a human patient. Methods to quantify inhibition are known in the
art, see, U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467 and
U.S. Pat. No. 7,601,702. For example, a fixed dose of a dUTPase
directed therapy (e.g., 5-FU or Pemetrexed) can be added to the
system and varying amounts of the compound can be subsequently
added to system. Alternatively, a fixed dose of a compound of this
invention can be added to the system and varying amounts of the
dUTPase directed therapy (e.g., 5-FU or Pemetrexed) compound can be
subsequently added to system.
[0242] In another aspect, the assay requires contacting a first
sample comprising suitable cells or tissue ("control sample") with
an effective amount of a composition of this invention and
optionally a dUTPase inhibitor, and contacting a second sample of
the suitable cells or tissue ("test sample") with the agent to be
assayed and optionally a dUTPase inhibitor. In one aspect, the cell
or tissue over express dUTPase. The inhibition of growth of the
first and second cell samples are determined. If the inhibition of
growth of the second sample is substantially the same or greater
than the first sample, then the agent is a potential drug for
therapy. In one aspect, substantially the same or greater
inhibition of growth of the cells is a difference of less than
about 1%, or alternatively less than about 5% or alternatively less
than about 10%, or alternatively greater than about 10%, or
alternatively greater than about 20%, or alternatively greater than
about 50%, or alternatively greater than about 90%. The contacting
can be in vitro or in vivo. Means for determining the inhibition of
growth of the cells are well known in the art.
[0243] In a further aspect, the test agent is contacted with a
third sample of cells or tissue comprising normal counterpart cells
or tissue to the control (or alternatively cells that do not over
express dUTPase) and test samples and selecting agents that treat
the second sample of cells or tissue but does not adversely effect
the third sample. For the purpose of the assays described herein, a
suitable cell or tissue is described herein such as cancer or other
diseases as described herein. Examples of such include, but are not
limited to cancer cell or tissue obtained by biopsy, blood, breast
cells, colon cells.
[0244] Efficacy of the test composition is determined using methods
known in the art which include, but are not limited to cell
viability assays or apoptosis evaluation.
[0245] In yet a further aspect, the assay requires at least two
cell types, the first being a suitable control cell.
[0246] The assays also are useful to predict whether a subject will
be suitably treated by this invention by delivering a composition
to a sample containing the cell to be treated and assaying for
treatment which will vary with the pathology or for screening for
new drugs and combinations. In one aspect, the cell or tissue is
obtained from the subject or patient by biopsy. Applicants provide
kits for determining whether a pathological cell or a patient will
be suitably treated by this therapy by providing at least one
composition of this invention and instructions for use.
[0247] The test cells can be grown in small multi-well plates and
is used to detect the biological activity of test compounds. For
the purposes of this invention, the successful candidate drug will
block the growth or kill the pathogen but leave the control cell
type unharmed.
[0248] The following examples are included to demonstrate some
embodiments of the disclosure. However, those of skill in the art
should, in light of the present disclosure, appreciate that many
changes can be made in the specific embodiments which are disclosed
and still obtain a like or similar result without departing from
the spirit and scope of the invention.
Example 1
Synthesis of PCI 10213
##STR00050##
[0250] Piperidine-2,6-dione was treated with a suitable base such
as lithium hexamethyldisilazide, lithiumdiisopropylamide (LDA), or
LDA/hexamethylphosphoramide (HMPT) in tetrahydrofuran as a solvent.
It was then coupled with the bromide or chloride as shown in step 2
above, followed by acid hydrolysis to remove the sulfonamide
protecting group, to provide PCI 10213.
[0251] Other compounds of formula (I) and (III) were prepared in an
analogous manner. In some cases, protection of the "NH" group on
the piperidine-2,4-dione is required.
Example 2
Synthesis of PCI 10214
##STR00051##
[0253] Thiazolidine-2,4-dione was treated with a suitable base such
as n-butyl lithium, secondary butyllithium, or LDA/HMPT in a
solvent such as tetrahydrofuran or dimethylformamide. It was then
coupled with the bromide or chloride as shown in step 2 above,
followed by acid hydrolysis to remove the sulfonamide protecting
group, to provide PCI 10214.
[0254] Other compounds of formula (I) and (III) can be and were
prepared in an analogous manner. In some cases, protection of the
"NH" group on the thiazolidine-2,4-dione is required.
Example 3
Preparation of Stereochemically Pure Compounds
[0255] The disclosed compounds exist as two diastereomers differing
at only one single stereo center. This example demonstrates a
separation protocol. The stereochemical pure compounds were
prepared and then tested to determine if the biological activity is
attributed to one or both stereoisomers.
[0256] Separation of the diastereomers was performed by preparative
chiral high performance liquid chromatography (HPLC) employing a
250.times.30 mm CHIRALPAK IA (5 .mu.m) column, heptane/iso-propanol
(70/30) with a flow-rate of 42.5 mL/min and UV detection
(.lamda.=270 nm at 25.degree. C.). Analytical chiral HPLC was
performed employing a 250.times.4.6 mm CHIRALPAK IA (5 .mu.m)
column, heptane/iso-propanol/diethylamine (70/30/0.1) with a flow
rate of 1 mL/min and UV detection (1=230 nm at 25.degree. C.).
[0257] PCI 10213 exists as a mixture of diasteromers differing at
the chiral carbon shown in Example 1. PCI 10213 was separated by
preparative chiral HPLC under the above specified conditions to
provide enantiomers PCI 10586 and PCI 10585 that were in >99%
enantiomeric excess and >95% purity. FIGS. 9 and 10 show the
chiral HPLC chromatograms of PCI 10586 and PCI 10585 with retention
times (R.sub.t) of 28.4 and 22.13 mins, respectively.
Example 4
Key Intermediate I
(S)-1-azido-2-(3-(cyclopropylmethoxy)-4-fluorophenyl)butan-2-ol
[0258] Key intermediate I was prepared according to the literature
data (J. Med. Chem. 2012, 55, 6427).
General Procedure A
Alkylation with LiHMDS
##STR00052##
[0260] At -40.degree. C., a solution of lithium
bis(trimethylsilyl)amide 1 M in tetrahydrofuran (38.9 mmol, 38.9
mL, 2.2 eq) was added dropwise to a solution of glutarimide (2.0 g,
17.7 mmol, 1.0 eq) in tetrahydrofuran (30 mL). The iodoalkane (53.1
mmol, 3.0 eq) was immediately added. After 15 minutes at
-40.degree. C., the mixture was allowed to warm up and the mixture
was stirred at room temperature for 18 hours. The reaction was
quenched with a saturated solution of ammonium chloride (10 mL) and
the aqueous phase was extracted with methylene chloride (3.times.20
mL). The combined organic phases were dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The residue was
purified by flash chromatography using cyclohexane and ethyl
acetate (100/0 to 0/100) to afford the expected compound.
General Procedure B
Alkylation with LDA
##STR00053##
[0262] At 0.degree. C., a solution of lithium diisopropylamide 2 M
in tetrahydrofuran/heptane/ethylbenzene (38.9 mmol, 19.5 mL, 2.2
eq) was added dropwise to a solution of glutarimide (2.0 g, 17.7
mmol, 1.0 eq) in tetrahydrofuran (30 mL). The iodoalkane (53.1
mmol, 3.0 eq) was immediately added. After 15 minutes at 0.degree.
C., the mixture was allowed to warm up and then stirred at room
temperature for 18 hours. The reaction was quenched with water (10
mL) and the aqueous phase was extracted with methylene chloride
(3.times.20 mL). The combined organic phases were dried over
magnesium sulfate, filtered and evaporated under reduced pressure.
The residue was purified by flash chromatography using cyclohexane
and ethyl acetate (100/0 to 0/100) to afford the expected
compound.
General Procedure C
Reductive Amination
##STR00054##
[0264] To a solution of the amino compound (HCl Salt) (1.0 eq) in
methanol (10 mL) was added a 7 N solution of ammonia in methanol
(3.0 eq). The mixture was stirred at room temperature during 15
minutes and acetic acid was added until pH=5. The aldehyde (1.0 eq)
and sodium cyanoborohydride (3.0 eq) were added and the mixture was
stirred at room temperature for 18 hours. The reaction mixture was
carefully quenched with a saturated solution of sodium
hydrogenocarbonate (10 mL). The aqueous phase was extracted with
ethyl acetate (3.times.15 mL). The combined organic phases were
dried over magnesium sulfate, filtered and evaporated under reduced
pressure. The residue was purified by flash chromatography using
cyclohexane and ethyl acetate (100/0 to 0/100) to afford the
expected compound.
General Procedure D
"Click Chemistry"
##STR00055##
[0266] To a solution of the alkynyl compound (1.0 eq) and Key
Intermediate 1 (1.0 eq) in dioxane (10 mL) degazed with argon was
added
chloro(1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium II
(0.1 eq). The reaction mixture was stirred at 80.degree. C. for 3
hours. After cooling down, the reaction mixture was evaporated
under vacuum and the residue was absorbed on silica gel to be
purified by flash chromatography using cyclohexane and ethyl
acetate (100/0 to 0/100) to afford the expected compound.
Example 5
3-(4-(3-[(S)-2-(3-Cyclopropylmethoxy-4-fluorophenyl)-2-hydroxybutyl]-3H-[1-
,2,3]triazol-4-yl)-butyl)-piperidine-2,6-dione
##STR00056##
[0267] Step 1
[0268] 3-hex-5-ynyl-piperidine-2,6-dione was prepared according to
General Procedure A using glutarimide (2.0 g, 17.7 mmol) and
6-iodo-1-hexyne (5.6 mL, 42.4 mmol). The expected compound was
isolated as orange oil that solidified during storage with 17%
yield (570 mg).
Step 2
[0269] The title compound was prepared according to General
Procedure D, using 3-hex-5-ynyl-piperidine-2,6-dione prepared in
step 1 (173 mg, 0.9 mmol) and Key Intermediate I (250 mg, 0.9
mmol). The expected compound was isolated as beige foam with 69%
yield (291 mg).
[0270] .sup.1H NMR (CDCl.sub.3): 7.83 (broad s, 1H), 7.37 (s, 1H),
6.99 (ddd, J=1.5, 8.5 and 12.4 Hz, 1H), 6.89 (dd, J=2.2 and 8.2 Hz,
1H), 7.76 (m, 1H), 4.45 (d, J=14.0 Hz, 1H), 4.34 (d, J=14.0 Hz,
1H), 3.78 (d, J=7.0 Hz, 2H), 2.72 (m, 1H), 2.56 (m, 1H), 2.36 (m,
3H), 2.21-1.70 (m, 6H), 1.53 (m, 3H), 1.36 (m, 2H), 1.22 (m, 1H),
0.83 (t, J=7.3 Hz, 3H), 0.62 (m, 2H), 0.32 (m, 2H)
Example 6
3-(5-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-pentyl)-piperidine-2,6-dione
##STR00057##
[0271] Step 1
[0272] 3-hept-6-ynyl-piperidine-2,6-dione was prepared according to
General Procedure A using glutarimide (2.0 g, 17.7 mmol) and
7-iodo-hept-1-yne (9.4 g, 42.5 mmol). The expected compound was
isolated as beige powder with 29% yield (1.07 g).
Step 2
[0273] The title compound was prepared according to General
Procedure D, using 3-hept-6-ynyl-piperidine-2,6-dione prepared in
step 1 (185 mg, 0.9 mmol) and Key Intermediate I (250 mg, 0.9
mmol). The expected compound was isolated as solidified oil with
67% yield (290 mg).
[0274] .sup.1H NMR (CDCl.sub.3): 7.80 (broad s, 1H), 7.36 (s, 1H),
6.99 (dd, J=8.5 and 10.8 Hz, 1H), 6.90 (m, 1H), 6.77 (m, 1H), 4.45
(d, J=14.0 Hz, 1H), 4.34 (dd, J=1.5 and 14.0 Hz, 1H), 3.78 (d,
J=7.0 Hz, 2H), 2.72 (dt, J=4.8 and 17.7 Hz, 1H), 2.55 (m, 1H), 2.42
(m, 1H), 2.27 (m, 2H), 2.12-1.69 (m, 6H), 1.61-1.19 (m, 8H), 0.82
(t, J=7.3 Hz, 3H), 0.62 (m, 2H), 0.33 (m, 2H)
Example 7
3-(3-(3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl)-propyl)-piperidine-2,6-dione
##STR00058##
[0275] Step 1
[0276] 3-pent-4-ynyl-piperidine-2,6-dione was prepared according to
General Procedure A using glutarimide (1.0 g, 8.8 mmol) and
5-iodo-pent-1-yne (5.0 g, 25.6 mmol). The expected compound was
isolated as white powder with 10% yield (152 mg).
Step 2
[0277] The title compound was prepared according to General
Procedure D, using 3-pent-4-ynyl-piperidine-2,6-dione prepared in
step 1 (150 mg, 0.8 mmol) and Key Intermediate I (234 mg, 0.8
mmol). The expected compound was isolated as white powder with 64%
yield (246 mg) after purification and lyophilization.
[0278] .sup.1H NMR (DMSO): 10.58 (s, 1H), 7.39 (s, 1H), 7.06 (dd,
J=8.5 and 11.3 Hz, 1H), 6.93 (dd, J=1.9 and 8.4 Hz, 1H), 6.82 (m,
1H), 5.28 (s, 1H), 4.40 (s, 2H), 3.76 (d, J=7.0 Hz, 2H), 2.60-2.20
(m, 5H), 1.92 (m, 2H), 1.75 (m, 2H), 1.51 (m, 3H), 1.35 (m, 1H),
1.16 (m, 1H), 0.66 (t, J=7.2 Hz, 3H), 0.53 (m, 2H), 0.29 (m,
2H)
Example 8
3-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butylamino)-piperidine-2,6-dione
##STR00059##
[0279] Step 1
[0280] 3-hex-5-ynylamino-piperidine-2,6-dione was prepared
according to General Procedure C using 3-aminopiperidine-2,6-dione
hydrochloride (500 mg, 3.0 mmol) and hex-5-ynal (292 mg, 3.0 mmol)
prepared from hex-5-yn-1-ol according to the procedure described in
the literature (US2011/306551). Before addition of the solution of
sodium hydrogenocarbonate (10 mL), the reaction mixture was
concentrated. The expected compound was isolated with 32% yield
(203 mg).
Step 2
[0281] To a solution of 3-hex-5-ynylamino-piperidine-2,6-dione
prepared in step 1 (188 mg, 0.9 mmol, 1.0 eq) in acetonitrile (15
mL) were added di-tert-butyl dicarbonate (433 mg, 1.98 mmol, 2.2
eq) and 4-dimethylaminopyridine (11 mg, 0.09 mmol, 0.1 eq). The
mixture was stirred at room temperature during 18 hours. The
reaction was quenched with a saturated solution of sodium
hydrogenocarbonate (10 mL) and extracted with ethyl acetate
(3.times.15 mL). The combined organic phases were dried over
magnesium sulfate, filtered and evaporated under reduced pressure.
The residue was purified by flash chromatography using cyclohexane
and ethyl acetate (100/0 to 60/40) to afford
(2,6-dioxo-piperidin-3-yl)-hex-5-ynyl-carbamic acid tert-butyl
ester with 50% yield (139 mg).
Step 3
[0282]
(4-{3-[(S)-2-(3-cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl-
]-3H-[1,2,3]triazol-4-yl}-butyl)-(2,6-dioxo-piperidin-3-yl)-carbamic
acid tert-butyl ester was prepared according to General Procedure
D, using (2,6-dioxo-piperidin-3-yl)-hex-5-ynyl-carbamic acid
tert-butyl ester prepared in step 2 (125 mg, 0.4 mmol) and Key
Intermediate I (113 mg, 0.4 mmol). The expected compound was
obtained as beige foam with 68% yield (160 mg).
Step 4
[0283] To a solution of
(4-{3-[(S)-2-(3-cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[-
1,2,3]triazol-4-yl}-butyl)-(2,6-dioxo-piperidin-3-yl)-carbamic acid
tert-butyl ester prepared in step 3 (160 mg, 0.3 mmol, 1.0 eq) in
methylene chloride (10 mL) was added a 1 M solution of
hydrochloride in diethyl ether (10 mL). After stirring at room
temperature during 3 hours, the mixture was concentrated and a
saturated solution of sodium hydrogenocarbonate (15 mL) was added.
The aqueous phase was extracted with ethyl acetate (3.times.15 mL).
The combined organic phases were dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The residue was
purified by flash chromatography using ethyl acetate and methanol
(100/0 to 80/20) and lyophilized to afford the expected compound as
white solid with 54% yield (71 mg).
[0284] .sup.1H NMR (DMSO): 10.66 (s, 1H), 7.36 (s, 1H), 7.06 (dd,
J=8.5 and 11.3 Hz, 1H), 6.92 (dd, J=2.1 and 8.4 Hz, 1H), 6.82 (m,
1H), 5.27 (s, 1H), 4.40 (s, 2H), 3.76 (d, J=7.0 Hz, 2H), 3.26 (m,
1H), 2.65-2.40 (m, 3H), 2.25 (m, 4H), 1.98 (m, 2H), 1.74 (m, 2H),
1.40 (m, 4H), 1.17 (m, 1H), 0.66 (t, J=7.2 Hz, 3H), 0.55 (m, 2H),
0.30 (m, 2H)
Example 9
3-(3-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-propylamino)-piperidine-2,6-dione
##STR00060##
[0285] Step 1
[0286] 3-pent-4-ynylamino-piperidine-2,6-dione was prepared
according to General Procedure C using 3-aminopiperidine-2,6-dione
hydrochloride (800 mg, 4.9 mmol) and pent-4-ynal (1.5 g, 18.8 mmol)
prepared from pent-5-yn-1-ol according to the procedure described
in the literature (US2011/306551). Before addition of the solution
of sodium hydrogenocarbonate (10 mL), the reaction mixture was
evaporated. The expected compound was isolated with 21% yield (200
mg).
Step 2
[0287] To a solution of 3-pent-4-ynylamino-piperidine-2,6-dione
prepared in step 1 (190 mg, 1.0 mmol, 1.0 eq) in acetonitrile (15
mL) were added di-tert-butyl dicarbonate (470 mg, 2.2 mmol, 2.2 eq)
and 4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.1 eq). The mixture
was stirred at room temperature for 18 hours. The reaction was
quenched with a saturated solution of sodium hydrogenocarbonate (10
mL) and extracted with ethyl acetate (3.times.15 mL). The combined
organic phases were dried over magnesium sulfate, filtered and
evaporated under reduced pressure. The residue was purified by
flash chromatography using cyclohexane and ethyl acetate (100/0 to
60/40) to afford (2,6-dioxo-piperidin-3-yl)-pent-4-ynyl-carbamic
acid tert-butyl ester with 60% yield (180 mg).
Step 3
[0288]
(3-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl-
]-3H-[1,2,3]triazol-4-yl}-propyl)-(2,6-dioxo-piperidin-3-yl)-carbamic
acid tert-butyl ester was prepared according to General Procedure
D, using (2,6-dioxo-piperidin-3-yl)-pent-4-ynyl-carbamic acid
tert-butyl ester prepared in step 2 (180 mg, 0.6 mmol, 1.0 eq) and
Key Intermediate I (171 mg, 0.6 mmol, 1.0 eq). The compound was
obtained as beige foam with 49% yield (170 mg).
Step 4
[0289] To a solution of
(3-{3-[(S)-2-(3-cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[-
1,2,3]triazol-4-yl}-propyl)-(2,6-dioxo-piperidin-3-yl)-carbamic
acid tert-butyl ester prepared in step 3 (170 mg, 0.3 mmol, 1.0 eq)
in methylene chloride (10 mL) was added a 1 M solution of
hydrochloride in diethyl ether (10 mL). After stirring at room
temperature during 3 hours, the mixture was concentrated and a
saturated solution of sodium hydrogenocarbonate (15 mL) was added.
The aqueous phase was extracted with ethyl acetate (3.times.15 mL).
The combined organic phases were dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The residue was
purified by flash chromatography using ethyl acetate and methanol
(100/0 to 90/10) and lyophilized to afford the title compound with
88% yield as light blue solid (125 mg).
[0290] .sup.1H NMR (DMSO): 10.66 (s, 1H), 7.38 (s, 1H), 7.06 (dd,
J=8.4 and 11.0 Hz, 1H), 6.93 (d, J=8.4 Hz, 1H), 6.83 (m, 1H), 5.27
(s, 1H), 4.41 (s, 2H), 3.77 (d, J=7.0 Hz, 2H), 2.25 (m, 1H), 2.57
(m, 3H), 2.36 (m, 3H), 2.21 (m, 1H), 1.96 (m, 2H), 1.82-1.50 (m,
4H), 1.16 (m, 1H), 0.66 (t, J=7.2 Hz, 3H), 0.55 (m, 2H), 0.29 (m,
2H)
Example 10
3-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluorophenyl)-2-hydroxybutyl]-3H-[1-
,2,3]triazol-4-yl}-butylamino)-3-methyl-piperidine-2,6-dione
##STR00061##
[0291] Step 1
[0292] 3-hex-5-ynylamino-3-methyl-piperidine-2,6-dione was prepared
according to General Procedure C using
3-amino-3-methyl-piperidine-2,6-dione hydrochloride monohydrate
prepared according the procedure described in the literature
(WO2006/081251) (600 mg, 3.0 mmol) and hex-5-ynal (440 mg, 3.0
mmol) prepared from hex-5-yn-1-ol according to the procedure
described in the literature (US2011/306551). The expected compound
was isolated with 41% yield (280 mg).
Step 2
[0293] The title compound was prepared according to General
Procedure D, using 3-hex-5-ynylamino-3-methyl-piperidine-2,6-dione
prepared in step 1 (100 mg, 0.4 mmol) and Key Intermediate I (126
mg, 0.4 mmol). The expected compound was obtained as white powder
after purification and lyophilization with 29% yield (65 mg).
[0294] .sup.1H NMR (DMSO): 10.56 (s, 1H), 7.37 (s, 1H), 7.08 (dd,
J=8.5 and 11.3 Hz, 1H), 6.93 (dd, J=1.9 and 8.5 Hz, 1H), 6.85 (m,
1H), 5.29 (s, 1H), 4.41 (s, 2H), 3.78 (d, J=7.0 Hz, 2H), 2.63 (m,
1H), 2.34 (m, 5H), 1.99 (m, 3H), 1.76 (m, 2H), 1.43 (m, 2H), 1.31
(m, 2H), 1.18 (m, 4H), 0.68 (t, J=7.2 Hz, 3H), 0.56 (m, 2H), 0.31
(m, 2H)
Example 11
3-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
##STR00062##
[0295] Step 1
[0296] 3-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one was
prepared according to General Procedure B using
3,4-dihydro-1H-[1,8]naphthyridin-2-one (300 mg, 2.0 mmol) and
6-iodo-1-hexyne (790 .mu.L, 6.0 mmol). The expected compound was
isolated as yellow powder with 13% yield.
Step 2
[0297] The title compound was prepared according to General
Procedure D, using
3-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in
step 1 (60 mg, 0.3 mmol) and Key Intermediate I (73 mg, 0.3 mmol).
The expected compound was isolated as white powder after flash
chromatography and lyophilization with 55% yield (73 mg).
[0298] .sup.1H NMR (CDCl.sub.3): 8.80 (broad s, 1H), 8.18 (d, J=4.3
Hz, 1H), 7.56 (d, J=7.3 Hz, 1H), 7.33 (s, 1H), 6.99 (m, 2H), 6.89
(m, 1H), 6.77 (m, 1H), 4.43 (dd, J=1.7 and 14.0 Hz, 1H), 4.33 (d,
J=14.0 Hz, 2H), 3.77 (dd, J=1.9 and 7.0 Hz, 2H), 3.03 (dd, J=6.0
and 16.0 Hz, 1H), 2.74 (m, 1H), 2.57 (m, 1H), 2.28 (m, 2H), 2.00
(m, 1H), 1.84 (m, 2H), 1.60-1.30 (m, 5H), 1.21 (m, 1H), 0.83 (t,
J=7.4 Hz, 3H), 0.61 (m, 2H), 0.31 (m, 2H)
Example 12
3-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butyl)-8-methoxy-3,4-dihydro-1H-quinolin-2-one
##STR00063##
[0299] Step 1
[0300] To a solution of 2-chloro-8-methoxy-quinoline (2.1 g, 10.7
mmol, 1.0 eq) in acetic acid (15 mL) was added water (5 mL). The
mixture was stirred at 100.degree. C. during 18 hours. After
cooling down, the solvent was evaporated. Water (30 mL) and a 25%
solution of ammonium hydroxide (20 mL) were added. The aqueous
phase was extracted with methylene chloride (2.times.20 mL) and
chloroform (20 mL). The combined organic phases were dried over
magnesium sulfate, filtered and evaporated under reduced pressure
to afford 8-methoxy-1H-quinolin-2-one as white powder with
quantitative yield (1.9 g).
Step 2
[0301] To a solution of 8-methoxy-1H-quinolin-2-one prepared in
step 1 (430 mg, 2.4 mmol, 1.0 eq) in ethanol (40 mL) was added
rhodium on alumina powder. The suspension was hydrogenated under 4
bars of dihydrogen for 4 hours at 30.degree. C. Then, the
suspension was filtered over celite and evaporated under vacuum to
afford a 70/30 mixture of 8-methoxy-3,4-dihydro-1H-quinolin-2-one
and starting material. The mixture (430 mg) was used crude in the
next step without purification.
Step 3
[0302] 3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one was
prepared according to General Procedure B using
8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in step 2 (430 mg,
2.4 mmol) and 6-iodo-1-hexyne (960 .mu.L, 7.3 mmol). The expected
compound was isolated as light yellow powder (231 mg).
Step 4
[0303] The title compound was prepared according to General
Procedure D, using
3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in
step 3 (100 mg, 0.4 mmol) and Key Intermediate I (119 mg, 0.4
mmol). The expected compound was isolated as beige powder after
flash chromatography and lyophilization with 69% yield (145
mg).
[0304] .sup.1H NMR (DMSO): 8.97 (s, 1H), 7.36 (s, 1H), 7.05 (dd,
J=8.5 and 11.3 Hz, 1H), 6.92-6.74 (m, 5H), 5.27 (s, 1H), 4.39 (s,
2H), 3.75 (m, 5H), 2.92 (dd, J=5.7 and 15.6 Hz, 1H), 2.63 (m, 1H),
2.32 (m, 3H), 1.98 (m, 1H), 1.75 (m, 1H), 1.63 (m, 1H), 1.41 (m,
2H), 1.28 (m, 3H), 1.15 (m, 1H), 0.66 (t, J=7.2 Hz, 3H), 0.53 (m,
2H), 0.28 (m, 2H)
Example 13
2-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butyl)-4H-pyrido[3,2-b][1,4]oxazin-3-one
##STR00064##
[0305] Step 1
[0306] To a stirred solution of trimethylsilylacetylene (595 .mu.L,
4.2 mmol, 3.0 eq) in dry tetrahydrofurane was added at -78.degree.
C. a 2 M solution of n-butyllithium in hexane (2.4 mL, 4.9 mmol,
3.5 eq). After 2 minutes, hexamethylphosphoramide (0.64 mL) and
2-(4-bromo-butyl)-4H-pyrido[3,2-b][1,4]oxazin-3-one (400 mg, 1.4
mmol, 1.0 eq) were added. The mixture was stirred from -78.degree.
C. to room temperature during 18 hours. The mixture was then
quenched with water (10 mL) and extracted with methylene chloride
(3.times.10 mL). The combined organic phases were dried over
magnesium sulfate, filtered and evaporated under reduced pressure.
The LC/MS analysis of the orange oil obtained showed a mixture of
sillylated compound and terminal alkyne.
[0307] The mixture was solubilized in THF and a 1 M solution of
tetrabutylammonium fluoride in tetrahydrofurane (2.8 mL, 2.8 mmol,
2.0 eq) was added. The mixture was stirred at room temperature for
18 hours. Water (10 mL) was added and the mixture was extracted
with ethyl acetate (3.times.10 mL). The combined organic phases
were dried over magnesium sulfate, filtered and evaporated under
reduced pressure. The residue was purified by flash chromatography
using cyclohexane and ethyl acetate (100/0 to 80/20) to afford
2-hex-5-ynyl-4H-pyrido[3,2-b][1,4]oxazin-3-one with an overall
yield of 19% (60 mg).
Step 2
[0308] The expected compound was prepared according to General
Procedure D, using 2-hex-5-ynyl-4H-pyrido[3,2-b][1,4]oxazin-3-one
prepared in step 1 (60 mg, 0.3 mmol) and Key Intermediate 1 (73 mg,
0.3 mmol). After the flash chromatography, the compound was
purified by preparative HPLC to afford after lyophilization the
expected compound as light blue solid with 38% yield (51 mg).
[0309] .sup.1H NMR (CDCl.sub.1): 7.98 (d, 4.5 Hz, 1H), 7.35 (s,
1H), 7.32 (d, J=8.0 Hz, 1H), 6.98 (m, 2H), 6.91 (dd, J=2.2 and 8.1
Hz, 1H), 6.76 (m, 1H), 4.62 (m, 1H), 4.44 (dd, J=2.1 and 14.0 Hz,
1H), 4.33 (d, J=14.0 Hz, 1H), 3.78 (d, J=7.0 Hz, 2H), 2.31 (m, 2H),
1.97 (m, 3H), 1.81 (m, 1H), 1.54 (m, 4H), 1.22 (m, 2H), 0.82 (t,
J=7.3 Hz, 3H), 0.63 (m, 2H), 0.32 (m, 2H)
Example 14
6-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
##STR00065##
[0310] Step 1
[0311] In a sealed tube, to a suspension of
6-bromo-3,4-dihydro-1H-[1,8]naphthyridin-2-one (800 mg, 3.5 mmol,
1.0 eq) in toluene (20 mL) were added successively sodium carbonate
(746 mg, 7.0 mmol, 2.0 eq), tributyl(vinyl)tin (1.3 g, 4.2 mmol,
1.2 eq) and water (1 mL). The suspension was degazed with argon and
tetrakis(triphenylphosphine)palladium(0) (407 mg, 0.3 mmol, 0.1 eq)
was added. The reaction mixture was stirred at 120.degree. C.
during 12 hours. After cooling down, a saturated solution of sodium
hydrogenocarbonate (10 mL) was added and the mixture was extracted
with ethyl acetate (2.times.10 mL) and methylene chloride (10 mL).
The combined organic phases were dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The crude residue
was purified by flash chromatography using cyclohexane and ethyl
acetate (100/0 to 50/50). The residue obtained after solvent
evaporation was precipitated in methylene chloride and n-pentane to
afford 6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white
powder with 75% yield (460 mg).
Step 2
[0312] To a solution of
6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in step 1
(310 mg, 1.8 mmol, 1.0 eq) in methylene chloride (15 mL) was added
4-bromo-1-butene (360 .mu.L, 3.6 mmol, 2.0 eq). The solution was
degassed with argon before the addition of Grubbs' catalyst (second
generation) (75 mg, 0.09 mmol, 0.05 eq). The reaction mixture was
heated at 50.degree. C. during 4 hours. After cooling down, a
saturated solution of sodium hydrogenocarbonate (10 mL) was added
and the aqueous phase was extracted with methylene chloride (15 mL)
and ethyl acetate (2.times.15 mL). The combined organic phases were
dried over magnesium sulfate, filtered and evaporated under reduced
pressure. The crude residue was purified by flash chromatography
using cyclohexane and ethyl acetate (100/0 to 50/50) to afford
6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
as white powder with 66% yield (330 mg).
Step 3
[0313] To a solution of
6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
prepared in step 2 (330 mg, 1.2 mmol, 1.0 eq) in ethanol (40 mL)
was added rhodium on alumina powder. The suspension was
hydrogenated under 1 bar of dihydrogen during 4 hours at 15.degree.
C. Then, the suspension was filtered over celite and evaporated
under vacuum. The crude residue was purified by flash
chromatography using cyclohexane and ethyl acetate (100/0 to 50/50)
to afford 6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
as white powder with 54% yield (220 mg).
Step 4
[0314] To a solution of trimethylsilylacetylene (1.1 .mu.L, 7.8
mmol, 10.0 eq) in tetrahydrofuran (10 mL) was added at -78.degree.
C. a 2 M solution of n-butyllithium in cyclohexane (1.6 mL, 3.1
mmol, 4.0 eq) and HMPA (0.5 mL, 3.1 mmol, 4.0 eq). After 5 minutes,
a solution of
6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared
in step 3 (220 mg, 0.8 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was
added at -78.degree. C. The reaction mixture was stirred from
-78.degree. C. to room temperature for 18 hours. A saturated
solution of sodium hydrogenocarbonate (15 mL) was added and the
aqueous phase was extracted with ethyl acetate (3.times.15 mL). The
combined organic phases were dried over magnesium sulfate, filtered
and evaporated under reduced pressure. The crude residue was
purified by flash chromatography using cyclohexane and ethyl
acetate (100/0 to 50/50) to afford
6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
as white powder (210 mg) in mixture with traces of HMPA. This
mixture was used in the next step.
Step 5
[0315] To a solution of
6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
(210 mg, 0.7 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was added a
1M solution of tetra-n-butylammonium fluoride in tetrahydrofuran
(2.1 mL, 2.1 mmol, 3.0 eq). After 12 hours at room temperature,
water (10 mL) was added and the reaction mixture was extracted with
ethyl acetate (3.times.10 mL). The combined organic phases were
dried over magnesium sulfate, filtered and evaporated under reduced
pressure. The crude residue was purified by flash chromatography
using cyclohexane and ethyl acetate (100/0 to 40/60) to afford
6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white powder
(100 mg) with an overall yield of 56% on step 4 and step 5.
Step 6
[0316] The title compound was prepared according to General
Procedure D, using
6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in
step 5 (100 mg, 0.4 mmol) and Key Intermediate I (122 mg, 0.4
mmol). The expected compound was isolated as white powder after
flash chromatography and lyophilization with 56% yield (125
mg).
[0317] .sup.1H NMR (DMSO): 10.30 (s, 1H), 7.89 (d, J=2.0 Hz, 1H),
7.39 (s, 1H), 7.35 (s, 1H), 7.06 (dd, J=8.5 and 11.4 Hz, 1H), 6.92
(dd, J=2.0 and 8.5 Hz, 1H), 6.79 (m, 1H), 5.28 (s, 1H), 4.39 (s,
2H), 3.75 (d, J=7.0 Hz, 2H), 2.82 (t, J=7.5 Hz, 2H), 2.45 (m, 4H),
2.32 (m, 2H), 1.98 (m, 1H), 1.76 (m, 1H), 1.44 (m, 4H), 1.14 (m,
1H), 0.67 (t, J=7.2 Hz, 3H), 0.52 (m, 2H), 0.26 (m, 2H).
Example 15
6-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
##STR00066##
[0318] Step 1
[0319] In a sealed tube, to a suspension of
6-bromo-3,4-dihydro-1H-[1,8]naphthyridin-2-one (800 mg, 3.5 mmol,
1.0 eq) in toluene (20 mL) were added successively sodium carbonate
(746 mg, 7.0 mmol, 2.0 eq), tributyl(vinyl)tin (1.3 g, 4.2 mmol,
1.2 eq) and water (1 mL). The suspension was degazed with argon and
tetrakis(triphenylphosphine)palladium(0) (407 mg, 0.3 mmol, 0.1 eq)
was added. The reaction mixture was stirred at 120.degree. C.
during 12 hours. After cooling down, a saturated solution of sodium
hydrogenocarbonate (10 mL) was added and the mixture was extracted
with ethyl acetate (2.times.10 mL) and methylene chloride (10 mL).
The combined organic phases were dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The crude residue
was purified by flash chromatography using cyclohexane and ethyl
acetate (100/0 to 50/50). The residue obtained after solvent
evaporation was precipitated in methylene chloride and n-pentane to
afford 6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white
powder with 75% yield (460 mg).
Step 2
[0320] To a solution of
6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in step 1
(310 mg, 1.8 mmol, 1.0 eq) in methylene chloride (15 mL) was added
4-bromo-1-butene (360 .mu.L, 3.6 mmol, 2.0 eq). The solution was
degased with argon before the addition of Grubbs' catalyst (second
generation) (75 mg, 0.09 mmol, 0.05 eq). The reaction mixture was
heated at 50.degree. C. during 4 hours. After cooling down, a
saturated solution of sodium hydrogenocarbonate (10 mL) was added
and the aqueous phase was extracted with methylene chloride (15 mL)
and ethyl acetate (2.times.15 mL). The combined organic phases were
dried over magnesium sulfate, filtered and evaporated under reduced
pressure. The crude residue was purified by flash chromatography
using cyclohexane and ethyl acetate (100/0 to 50/50) to afford
6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
as white powder with 66% yield (330 mg).
Step 3
[0321] To a solution of
6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
prepared in step 2 (330 mg, 1.2 mmol, 1.0 eq) in ethanol (40 mL)
was added rhodium on alumina powder. The suspension was
hydrogenated under 1 bar of dihydrogen during 4 hours at 15.degree.
C. Then, the suspension was filtered over celite and evaporated
under vacuum. The crude residue was purified by flash
chromatography using cyclohexane and ethyl acetate (100/0 to 50/50)
to afford 6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
as white powder with 54% yield (220 mg).
Step 4
[0322] To a solution of trimethylsilylacetylene (1.1 .mu.L, 7.8
mmol, 10.0 eq) in tetrahydrofuran (10 mL) was added at -78.degree.
C. a 2 M solution of n-butyllithium in cyclohexane (1.6 mL, 3.1
mmol, 4.0 eq) and HMPA (0.5 mL, 3.1 mmol, 4.0 eq). After 5 minutes,
a solution of
6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared
in step 3 (220 mg, 0.8 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was
added at -78.degree. C. The reaction mixture was stirred from
-78.degree. C. to room temperature for 18 hours. A saturated
solution of sodium hydrogenocarbonate (15 mL) was added and the
aqueous phase was extracted with ethyl acetate (3.times.15 mL). The
combined organic phases were dried over magnesium sulfate, filtered
and evaporated under reduced pressure. The crude residue was
purified by flash chromatography using cyclohexane and ethyl
acetate (100/0 to 50/50) to afford
6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
as white powder (210 mg) in mixture with traces of HMPA. This
mixture was used in the next step.
Step 5
[0323] To a solution of
6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one
(210 mg, 0.7 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was added a
1M solution of tetra-n-butylammonium fluoride in tetrahydrofuran
(2.1 mL, 2.1 mmol, 3.0 eq). After 12 hours at room temperature,
water (10 mL) was added and the reaction mixture was extracted with
ethyl acetate (3.times.10 mL). The combined organic phases were
dried over magnesium sulfate, filtered and evaporated under reduced
pressure. The crude residue was purified by flash chromatography
using cyclohexane and ethyl acetate (100/0 to 40/60) to afford
6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white powder
(100 mg) with an overall yield of 56% on step 4 and step 5.
Step 6
[0324] The title compound was prepared according to General
Procedure D, using
6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in
step 5 (100 mg, 0.4 mmol) and Key Intermediate I (122 mg, 0.4
mmol). The expected compound was isolated as white powder after
flash chromatography and lyophilization with 56% yield (125
mg).
[0325] .sup.1H NMR (DMSO): 10.30 (s, 1H), 7.89 (d, J=2.0 Hz, 1H),
7.39 (s, 1H), 7.35 (s, 1H), 7.06 (dd, J=8.5 and 11.4 Hz, 1H), 6.92
(dd, J=2.0 and 8.5 Hz, 1H), 6.79 (m, 1H), 5.28 (s, 1H), 4.39 (s,
2H), 3.75 (d, J=7.0 Hz, 2H), 2.82 (t, J=7.5 Hz, 2H), 2.45 (m, 4H),
2.32 (m, 2H), 1.98 (m, 1H), 1.76 (m, 1H), 1.44 (m, 4H), 1.14 (m,
1H), 0.67 (t, J=7.2 Hz, 3H), 0.52 (m, 2H), 0.26 (m, 2H)
Example 16
3-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butyl)-8-methoxy-3,4-dihydro-1H-quinolin-2-one
##STR00067##
[0326] Step 1
[0327] To a solution of 2-chloro-8-methoxy-quinoline (2.1 g, 10.7
mmol, 1.0 eq) in acetic acid (15 mL) was added water (5 mL). The
mixture was stirred at 100.degree. C. during 18 hours. After
cooling down, the solvent was evaporated. Water (30 mL) and a 25%
solution of ammonium hydroxide (20 mL) were added. The aqueous
phase was extracted with methylene chloride (2.times.20 mL) and
chloroform (20 mL). The combined organic phases were dried over
magnesium sulfate, filtered and evaporated under reduced pressure
to afford 8-methoxy-1H-quinolin-2-one as white powder with
quantitative yield (1.9 g).
Step 2
[0328] To a solution of 8-methoxy-1H-quinolin-2-one prepared in
step 1 (430 mg, 2.4 mmol, 1.0 eq) in ethanol (40 mL) was added
rhodium on alumina powder. The suspension was hydrogenated under 4
bars of dihydrogen for 4 hours at 30.degree. C. Then, the
suspension was filtered over celite and evaporated under vacuum to
afford a 70/30 mixture of 8-methoxy-3,4-dihydro-1H-quinolin-2-one
and starting material. The mixture (430 mg) was used crude in the
next step without purification.
Step 3
[0329] 3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one was
prepared according to General Procedure B using
8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in step 2 (430 mg,
2.4 mmol) and 6-iodo-1-hexyne (960 .mu.L, 7.3 mmol). The expected
compound was isolated as light yellow powder (231 mg).
Step 4
[0330] The title compound was prepared according to General
Procedure D, using
3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in
step 3 (100 mg, 0.4 mmol) and Key Intermediate I (119 mg, 0.4
mmol). The expected compound was isolated as beige powder after
flash chromatography and lyophilization with 69% yield (145
mg).
[0331] .sup.1H NMR (DMSO): 8.97 (s, 1H), 7.36 (s, 1H), 7.05 (dd,
J=8.5 and 11.3 Hz, 1H), 6.92-6.74 (m, 5H), 5.27 (s, 1H), 4.39 (s,
2H), 3.75 (m, 5H), 2.92 (dd, J=5.7 and 15.6 Hz, 1H), 2.63 (m, 1H),
2.32 (m, 3H), 1.98 (m, 1H), 1.75 (m, 1H), 1.63 (m, 1H), 1.41 (m,
2H), 1.28 (m, 3H), 1.15 (m, 1H), 0.66 (t, J=7.2 Hz, 3H), 0.53 (m,
2H), 0.28 (m, 2H)
Example 17
3-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H--
[1,2,3]triazol-4-yl}-butyl)-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one
##STR00068##
[0332] Step 1
[0333] The 3-hex-5-ynyl-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one was
prepared according to General Procedure B using
1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one (350 mg, 2.6 mmol, 1.0 eq)
and 6-iodo-1-hexyne (310 .mu.L, 2.3 mmol, 0.9 eq). The expected
compound was isolated as white powder with 18% yield (100 mg).
Step 2
[0334] The expected compound was prepared according to General
Procedure D, using
3-hex-5-ynyl-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one prepared in
step 1 (100 mg, 0.5 mmol) and Key Intermediate I (130 mg, 0.5
mmol). The expected compound was isolated as white powder after
flash chromatography and lyophilization with 33% yield (77 mg).
[0335] .sup.1H NMR (DMSO): 10.93 (s, 1H), 8.03 (d, J=4.5 Hz, 1H),
7.55 (d, J=7.0 Hz, 1H), 7.32 (s, 1H), 7.04 (dd, J=9.0 and 11.5 Hz,
1H), 6.93 (m, 2H), 6.82 (m, 1H), 5.25 (s, 1H), 4.38 (s, 2H), 3.75
(d, J=7.0 Hz, 2H), 3.50 (t, J=5.8 Hz, 1H), 2.30 (m, 2H), 2.00-1.65
(m, 4H), 1.40 (m, 2H), 1.16 (m, 3H), 0.65 (t, J=7.2 Hz, 3H), 0.53
(m, 2H), 0.28 (m, 2H).
Example 18
Biological Methods
A. Drugs, Reagents and Cell Lines
[0336] PCI 10213, 10214 and 10216 are suspended in DMSO at a
concentration of 100 mmol/L, fluorodeoxyuridine (FUdR) that can be
obtained from Sigma (St Louis, Mo.) and maintained in sterile
double-distilled water at stock concentrations of 50 mmol/L. PCI
10216 has the structure:
##STR00069##
[0337] Recombinant human deoxyuridine nucleotidohydrolase (dUTPase)
is expressed and purified as described in Ladner R D, Carr S A,
Huddleston M J, McNulty D E, Caradonna S J. J Biol Chem. 1996 Mar.
29; 271 (13):7752-7. All drugs stocks were aliquoted and diluted as
appropriate prior to use. The oligonucelotide primer, templates and
fluorophore- and quencher-labeled detection probes are synthesized
by Integrated DNA Technologies (Coralville, Iowa), subjected to
polyacrylamide gel electrophoresis purification and reconstituted
in Omnipur sterile nuclease-free water (EMD Chemicals USA,
Gibbstown N.J.) at a stock concentration of 100 .mu.mol/L. The two
non-emissive (dark) quenching molecules incorporated into the
detection probes include the Iowa black fluorescein quencher (IBFQ;
absorption max 531 nm) and ZEN (non-abbreviation; absorption max
532 nm). The fluorescent label utilized was 6-FAM
(5'-carboxyfluorescein; excitation max.=494 nm, emission max.=520
nm). Probes were further diluted to a working stock of 10 .mu.mol/L
and aliquoted to avoid repeated freeze/thaw cycles. AmpliTaq Gold
DNA Polymerase, GeneAmp 10.times.PCR Buffer 2, MgCl.sub.2 and
MicroAmp Optical 96-well Reaction Plates were purchased from
Applied Biosystems (Carlsbad, Calif.). dNTPs were purchased
individually at stock concentrations of 100 mmol/L from New England
Biolabs at HPLC-certified >99% purity (Ipswich, Mass.).
B. Assay Components, Instrumentation and Real-Time Fluorescence
Conditions
[0338] Reaction mixtures contained primer, probe and template at an
equimolar final concentration of 0.4 .mu.mol/L. Magnesium chloride
(MgCl.sub.2) was included at a final concentration of 2 mmol/L.
Non-limiting dNTPs were included in the reaction mix in excess at a
final concentration of 100 .mu.mol/L (dUTP/dTTP was excluded).
AmpliTaq Gold DNA polymerase was added at 0.875 U/reaction, 2.5
.mu.l of 10.times.PCR buffer 2 added and nuclease-free ddH.sub.2O
added to a final reaction volume of 25 .mu.l. For dUTP inhibition
analysis, the volume of ddH.sub.2O was further modified to
accommodate an additional 1 .mu.l of dUTPase (10 ng/.mu.l) and 1
.mu.l of inhibitor or DMSO control. Thermal profiling and
fluorescence detection was performed using the `isothermal` program
on board an Applied Biosystems 7500 Real-Time PCR System. For
analysis of dNTPs, the thermal profile consisted of an 8 min
37.degree. C. step followed by a 10 min 95.degree. C. step to
`hot-start` the Taq polymerase and a primer extension time of up to
30 min at 60.degree. C. depending on the application. Raw
fluorescence spectra for 6-FAM was measured using filter A at
specified time intervals to follow assay progression using Sequence
Detection Software (SDS Version 1.4, Applied Biosystems) and
exported and analyzed in Microsoft Excel (Microsoft, Redmond Wash.)
and Prism (GraphPad Software, La Jolla Calif.). In all cases,
fluorescence values for blank reactions (limiting dNTP omitted)
were subtracted to give normalized fluorescence units (NFU) to
account for background fluorescence.
C. MTS Growth Inhibition Assay
[0339] The Cell Titer.sup.96 AQueous MTS assay (Promega) was
carried out according to the manufacturers guidelines.
IC.sub.50(72h) values were calculated from sigmoidal-dose response
curves utilizing Prism (Graphpad, San Diego, Calif.). The
combination effect was determined by the combination index (CI)
method utilizing Calcusyn software (Biosoft, Ferguson, Mo.).
Fraction affected (FA) was calculated from the percent growth
inhibition: FA=(100-% growth inhibition)/100. CI values <1,
synergism; 1-1.2, additive and >1.2, antagonism.
D. Colony Formation Assay
[0340] Colony forming assay showing the ability of colon (SW620,
HCT116), non-small cell lung (A549, H460, H1299 and H358) and
breast (MCF7) cancer cells to survive and proliferate following
transient 24 hour exposure to single agent PCI 1013, FUdR and
combinations. Specifically, cells were seeded at densities between
50 and 100 cells/well in 24-well plates. Twenty-four hours later,
cells were treated with increasing concentrations of PCI 10213, a
fixed dose of FUdR and combinations of these. After 24 hours, drug
was removed, cells were rinsed and allowed to outgrow for 10-14
days. At the conclusion of the outgrowth, cells were fixed in 60%
ice cold methanol and stained with 0.1% crystal violet, scanned and
counted. Data is presented as percentage of untreated controls
(mean.+-.SD). Fraction affected and combination indexes were
calculated according to the method of Chou and Talalay where <1
is indicative of a synergistic drug interaction.
E. In Vivo Analysis
[0341] Xenograft experiments were conducted in male NU/NU nude mice
(Charles River, Wilmington, Mass.) that were 6-8 weeks old.
Subcutaneous A549 xenografts were established and allowed to grow
until they reached .about.50 mm.sup.3 (day 1). Animals were
randomized to treatment groups: vehicle, pemetrexed 50 mg/kg, PCI
10213 and combination of pemetrexed plus PCI 10213 (n=5, group).
Pemetrexed was administered at 50 mg/kg by intraperitoneal
injection every two days. PCI 10213 was administered at 75 mg/kg by
intraperitoneal injection every two days. The combination of
pemetrexed and PCI 10214 was administered by intraperitoneal
injection every two days. Two perpendicular diameters of tumors
were measured every 2 days with a digital caliper by the same
investigator. Tumor volume was calculated according to the
following formula: TV
(mm.sup.3)=(length[mm].times.(width[mm].sup.2)/2. Mice were
inspected everyday for overall health and bodyweight was measured
every 2 days as an index of toxicity. All animal protocols were
approved by the USC Institutional Animal Care and Use Committee
(IACUC).
Example 19
Identification of the dUTPase Inhibitor PCI 10213
TABLE-US-00001 [0342] TABLE 1 % Inhibition umol/L 10216 10213 83.3
95.4 86.1 41.7 97.2 73.8 20.8 101.1 64.5 10.4 95.9 60.3 5.2 84.6
40.9 2.6 76.6 31.6 1.3 60.4 28.0
[0343] PCI 10213 and reference compound 10216 were screened in a
fluorescence-based assay. The assay employs a DNA polymerase-based
approach utilizing an oligonucleotide template with 3 distinct
regions: a 3' primer binding region, a mid-template dUTP/thymidine
triphosphate (TTP) detection region and a 5' 6-Flavin adenine
mononucleotide (FAM)-reaction, the probe and primer hybridize to
the oligonucleotide template to form the template:primer:probe
complex. When Taq polymerase binds to the primer in the TPP complex
and dUTP is present, successful extension of the nascent strand
occurs and the inherent 5' to 3' exonuclease activity of Taq
polymerase cleaves and displaces the 6-FAM-labeled probe in a 5' to
3' direction, releasing the 6-FAM fluorophore from its proximity to
the three quenchers. This displacement effectively disrupts the
Forster resonance energy transfer (FRET) and the resulting
fluorescence detected upon excitation is directly proportional to
the amount of the dUTP available in the assay for incorporation
(FIG. 3). Conversely, when the dUTP is unavailable, exhausted, or
degraded by dUTPase and is no longer available for incorporation,
Taq polymerase stalls and extension delay and/or chain termination
of the nascent strand occurs. In this instance, probe
hydrolysis/degradation does not occur and the probe remains dark as
fluorescence remains quenched via FRET. Since fluorescence is
directly proportional to the concentration of dUTP, the assay was
easily modified to measure dUTP and the effects of inhibitors on
dUTP hydrolysis by the enzyme dUTPase. The template BHQ-DT6 (Black
Hole Quencher--Detection Template 6) for detecting up to 60 pmols
of dUTP was included for this application of the assay along with
50 pmols of dUTP and 5 ng of recombinant dUTPase. The reaction was
incubated at 37.degree. C. for 8 mins and terminated by a 10 min
incubation at 95.degree. C. to simultaneously inactivate dUTPase
and activate the hot-start Taq polymerase. The fluorescence
generated during the detection step is directly proportional to the
concentration of dUTP remaining after the 8 min incubation. The
concentration of dUTP at reaction termination and therefore
inhibition of dUTPase in the presence and absence of inhibitors and
appropriate dimethyl sulfoxide (DMSO) controls can be determined.
In preliminary dUTPase inhibition experiments PCI 10213 was
compared directly to PCI 10216 at a range of concentrations between
0 and 83 .mu.mol/L (Table 1, FIG. 3A). Inhibition of dUTPase
enzymatic activity at the maximum dose of 83 .mu.mol/L was
significant for both compounds at 95 and 86% for PCI 10216 and
10213 respectively. The level of inhibition at 1.3 .mu.mol/L was
60% and 28% respectively. The IC.sub.50 calculated in Prism for PCI
10216 was 0.8 .mu.mol/L and for PCI 10213 7.2 .mu.mol/L.
Example 20
PCI 10213 Shows Little to No Single Agent Activity in Contrast to
PCI 10216
[0344] PCI 10213, 10214 and 10216 were evaluated for their
antitumor activity in colorectal cancer cells using the MTS growth
inhibition assay.
[0345] HCT116 and SW620 cells were exposed to increasing
concentrations of each agent for 72 hours and growth inhibition was
directly compared to vehicle-treated controls. In HCT116 cells, PCI
10213 and PCI 10214 demonstrated little to no single agent activity
even up to the elevated concentration of 75 .mu.mol/L with a modest
decrease in growth of 28% observed with 100 .mu.mol/L PCI 10213. In
contrast, PCI 10216 demonstrated dose-dependent decreases in cell
proliferation detectable at concentrations as low as 6.25 .mu.mol/L
and culminating with a 67% reduction in proliferation with 100
.mu.mol/L.
[0346] In SW620 cells, neither PCI 10213 or 10214 had any single
agent activity up to 75 .mu.mol/L and only modest activity of
.about.30% at 100 .mu.mol/L. PCI 10216 demonstrated dose-dependent
decreases in proliferation up to 70% at 100 .mu.mol/L (FIG.
3B).
[0347] The NSCLC cell lines A549 and H1299 were exposed to
increasing concentrations of each agent for 72 hours and growth
inhibition was directly compared to vehicle-treated controls. In
A549 cells, PCI 10213 and PCI 10214 demonstrated modest single
agent activity with the elevated concentration of 75 and 100
.mu.mol/L showing modest decreases in growth of .about.25% at 100
.mu.mol/L PCI 10213 and 10214. PCI 10216 demonstrated similar
decreases in cell proliferation as 10213 and 10214 at lower doses,
but significantly more at the elevated doses with 30% and 55%
reductions in proliferation with 75 and 100 mol/L respectively. In
H1299 cells, PCI 10213, 10214 and 10216 had modest single agent
activity up to 12.5 .mu.mol/L with increased activity of up to
.about.40% at 100 .mu.mol/L. Of note, PCI 10213 demonstrated
greater growth inhibition at 100 .mu.mol/L than PCI 10216 with
decreases in cell proliferation of 40% and 30% respectively at 100
.mu.mol/L.
Example 21
PCI 10213 Demonstrates Synergy with 5-FU Through Increase Growth
Inhibition
[0348] MTS growth inhibition assays were performed to evaluate the
effectiveness of both PCI 10213 and reference compound PCI 10216
alone and in combination with the fluoropyrimidine thymidylate
synthase (TS) inhibitor 5-fluorouracil (5-FU) at inhibiting the
growth of colorectal (HCT116 and SW620) cell line models.
Increasing concentrations of 5-FU between 0 and 100 .mu.mol/L
demonstrated dose-dependent increases in growth inhibition in both
the colorectal cancer cell lines evaluated. Simultaneous treatment
with increasing concentrations of 5-FU and either PCI 10213 and
10216 at fixed concentrations of 25 .mu.mol/L resulted in additive
and synergistic increases in growth inhibition over the majority of
concentrations tested up to 25 .mu.mol/L 5-FU in both CRC cell
lines examined. Of note, PCI 10216 as a single agent used at 25
.mu.mol/L induced 30% growth inhibition in SW620 cells and 44% in
HCT116 cells whereas PCI 10213 had no detectable effect on growth
inhibition at 25 .mu.mol/L in either cell line despite showing
additive and synergistic interactions with 5-FU. These data
demonstrate a clear enhancement of 5-FU growth inhibitory activity
through the addition of PCI 10213 with significantly less single
agent activity than PCI 10216. See, FIG. 4.
Example 22
PCI 10213 Demonstrates Synergy with FUdR in Reducing Cancer Cell
Viability
[0349] Colony forming assays were performed to evaluate the
effectiveness of both PCI 10213, PCI 10214 and reference compound
PCI 10216 alone and in combination with the fluoropyrimidine
thymidylate synthase (TS) inhibitor fluorodeoxyuridine (FUdR) at
reducing cancer cell viability in colorectal (HCT116), breast
(MCF-7) and non-small cell lung (H1299, A549, H358 and H460) cell
line models. Increasing concentrations of FUdR between 0.5 and 2.5
.mu.mol/L demonstrated dose-dependent decreases in colonies formed
in all cell lines evaluated. Increasing concentrations of PCI 10213
between 3.1 and 25 .mu.mol/L had no significant effects on the
number of colonies formed whereas the elevated concentration of PCI
10216 at 25 and 50 .mu.mol/L demonstrated some reduction in the
number of colonies formed in A549, H460 and HCT116 cells. Reference
compound PCI 10216 demonstrated strong synergy when combined with
fixed doses of FUdR in all cell lines examined. Subsequently,
increasing concentrations of PCI 10213 were combined with a fixed
dose of FUdR to evaluate the combined drug effect. In NSCLC cells,
concentrations of PCI 10213 ranging from 3.1 .mu.mol/L to 25
.mu.mol/L were combined with 1 .mu.mol/L FUdR. One j.mu.mol/L FUdR
had no significant effect on number of colonies formed compared to
vehicle-treated controls. However, all combinations of PCI 10213
and 1 .mu.mol/L FUdR demonstrated highly significant reductions in
colonies formed when compared to the corresponding single agent
concentrations of PCI 10213 alone or 1 .mu.mol/L FUdR. The
effectiveness of this combination was pronounced in A549 and H460
cells where 12.5 and 25 .mu.mol/L PCI 10213 combined with 1
.mu.mol/L FUdR reduced cell viability by >95% compared to
vehicle-treated controls.
[0350] In colorectal cancer cells, concentrations of PCI 10213
ranging from 3.1 .mu.mol/L to 50 .mu.mol/L were combined with 0.5
.mu.mol/L FUdR in HCT116 cells and 1 .mu.mol/L FUdR in SW620
cells.
[0351] Similar to the NSCLC cells, neither PCI 10213 or the fixed
dose of FUdR single agent had any significant effect on the number
of colonies formed but demonstrated a strong synergistic reduction
in colonies in all combinations tested in HCT116 and SW620 cells.
Specifically, the combination of 12.5 and 25 .mu.mol/L PCI 10213
combined with 0.5 .mu.mol/L FUdR reduced colony formation by
>95% in HCT116 cells and >50% in the strongly FUdR-resistant
SW620 cell line. Importantly, despite neither agent alone exerting
any effect on the number of colonies formed, complete loss of
viability was achieved with the 50 .mu.mol/L PCI 10213 and FUdR
combination in HCT116 cells. In the MCF-7 breast cancer cell line,
treatment with 0.5 .mu.mol/L FUdR had no significant impact on the
number of colonies formed, but when combined with concentrations of
PCI 10213 ranging from 3.1 .mu.mol/L to 25 .mu.mol/L significant
reductions in colony formation of between 30 and 60% was observed.
See, FIGS. 5-7.
Example 23
Diastereomer PCI 10586 is the Primary Active Component of PCI
10213
A. Drugs and Reagents
[0352] PCI 10586, 10585 and 10213 were suspended in DMSO at a
concentration of 100 mmol/L, fluorodeoxyuridine (FUdR) was obtained
from Sigma (St Louis, Mo.) and maintained in sterile
double-distilled water at stock concentrations of 50 mmol/L.
Recombinant human deoxyuridine nucleotidohydrolase (dUTPase) was
expressed and purified as described previously [Please provide
reference or confirm as noted above]. All drugs stocks were
aliquoted and diluted as appropriate prior to use. The
oligonucelotide primer, templates and fluorophore- and
quencher-labeled detection probes were synthesized by Integrated
DNA Technologies (Coralville, Iowa), subjected to polyacrylamide
gel electrophoresis purification and reconstituted in Omnipur
sterile nuclease-free water (EMD Chemicals USA, Gibbstown N.J.) at
a stock concentration of 100 .mu.mol/L. The two non-emissive (dark)
quenching molecules incorporated into the detection probes include
the Iowa black fluorescein quencher (IBFQ; absorption max 531 nm)
and ZEN (non-abbreviation; absorption max 532 nm). The fluorescent
label utilized was 6-FAM (5'-carboxyfluorescein; excitation
max.=494 nm, emission max.=520 nm). Probes were further diluted to
a working stock of 10 .mu.mol/L and aliquoted to avoid repeated
freeze/thaw cycles. AmpliTaq Gold DNA Polymerase, GeneAmp
10.times.PCR Buffer 2, MgCl.sub.2 and MicroAmp Optical 96-well
Reaction Plates were purchased from Applied Biosystems (Carlsbad,
Calif.). dNTPs were purchased individually at stock concentrations
of 100 mmol/L from New England Biolabs at HPLC-certified >99%
purity (Ipswich, Mass.).
B Assay Components, Instrumentation and Real-Time Fluorescence
Conditions
[0353] Reaction mixtures contained primer, probe and template at an
equimolar final concentration of 0.4 .mu.mol/L. MgCl.sub.2 was
included at a final concentration of 3 mmol/L. Non-limiting dNTPs
were included in the reaction mix in excess at a final
concentration of 100 .mu.mol/L (dUTP/dTTP was excluded). AmpliTaq
Gold DNA polymerase was added at 0.875 U/reaction, 2.5 .mu.l of
10.times.PCR buffer 2 added and nuclease-free ddH.sub.2O added to a
final reaction volume of 30 .mu.l. For dUTP inhibition analysis,
the volume of ddH.sub.2O was further modified to accommodate an
additional 1 .mu.l of dUTPase (2.5 ng/.mu.l) and 1 .mu.l of
inhibitor or DMSO control. Thermal profiling and fluorescence
detection was performed using the `isothermal` program on board an
Applied Biosystems 7500 Real-Time PCR System. For analysis of
dNTPs, the thermal profile consisted of an 10 min 37.degree. C.
step followed by a 10 min 95.degree. C. step to `hot-start` the Taq
polymerase and a 5-cycle primer extension time of 10 min at
60.degree. C. Raw fluorescence spectra for 6-FAM was measured using
filter A at specified time intervals to follow assay progression
using Sequence Detection Software (SDS Version 1.4, Applied
Biosystems) and exported and analyzed in Microsoft Excel
(Microsoft, Redmond Wash.) and Prism (GraphPad Software, La Jolla
Calif.). In all cases, fluorescence values for blank reactions
(limiting dNTP omitted) were subtracted to give normalized
fluorescence units (NFU) to account for background
fluorescence.
C. dUTPase Inhibition Screening Reveals that Diastereomer PCI 10586
is the Primary Active Component of PCI 10213
[0354] PCI 10213 possesses two molecular diastereomers: PCI 10586
and 10585. The diastereomer compounds were isolated by preparative
chiral HPLC and screened in a novel fluorescence-based assay as
described in Wilson et al. (2011) Nucleic Acids Res., Sept. 1 39
(17). The assay employs a DNA polymerase-based approach utilizing
an oligonucleotide template with 3 distinct regions: a 3' primer
binding region, a mid-template dUTP/TTP detection region and a 5'
6-FAM-labeled probe binding region that incorporates a black hole
quenching moiety as previously described. Since fluorescence is
directly proportional to the concentration of dUTP, the assay was
easily modified to measure dUTP and the effects of inhibitors on
dUTP hydrolysis by the enzyme dUTPase. The template BHQ-DT6 for
detecting up to 60 pmols of dUTP was included for this application
of the assay along with 50 pmols of dUTP and 2.5 ng of recombinant
dUTPase. The reaction was incubated at 37.degree. C. for 10 mins
and terminated by a 10 min incubation at 95.degree. C. to
simultaneously inactivate dUTPase and activate the hot-start Taq
polymerase. The subsequent fluorescence detection step involved
five 10 min cycles at 60.degree. C. to completion. The fluorescence
generated during the detection step is directly proportional to the
concentration of dUTP remaining after the 10 min incubation. The
concentration of dUTP at reaction termination is directly
proportional to the extent of inhibition of dUTPase in the presence
and absence of inhibitors and appropriate DMSO controls.
TABLE-US-00002 TABLE 2 PCI 10586, PCI 10585, and PCI 10213 were
screened at the specified compound concentrations using a
fluorescence-based dUTPase inhibition assay to determine dUTPase
enzyme inhibition % dUTPase Inhibition .mu.mol/L PCI 10586 PCI
10585 PCI 10213 83.3 75.6 9.5 54.6 41.7 64.3 5.4 45.5 20.8 66.1 5.5
30.0 10.4 55.8 6.5 24.9 5.2 38.3 3.4 14.5 2.6 27.8 0.5 9.6 1.3 16.4
0.6 4.7
[0355] dUTPase inhibition comparisons were made between compounds
PCI 10213, 10585, and 10586 at a range of concentrations between
1.3 and 83.3 itmol/L (Table 2). Inhibition of dUTPase enzymatic
activity at the maximum dose of 83.3 .mu.mol/L was significant for
compound 10586 with 75.6% inhibition, moderate for compound 10213
with 54.6% inhibition and modest for compound 10585 with 9.5%. At
moderate concentration of 5.2 .mu.mol/L, compound 10586
demonstrated strong inhibition of 38%, compound 10213 had 14.5% and
10585 had 3.4% inhibition. The level of inhibition at 1.3 .mu.mol/L
was 16.4%, 4.7%, and 0.6% for 10585, 10213 and 10585 respectively.
The strong dUTPase inhibition observed for 10586, the intermediate
inhibition of the heterogeneous 10213 and the distinct lack of
dUTPase inhibition by 10585 confirms that diasteromer PCI 10586
(28.46 min retention time) is the primary active molecule in
compound PCI 10213.
D. PCI 10586 Demonstrated Synergy with FUdR
[0356] Colony forming assays were performed to evaluate the
effectiveness of both PCI 10213, PCI 10585 and PCI 10586 alone and
in combination with the fluoropyrimidine thymidylate synthase (TS)
inhibitor fluorodeoxyuridine (FUdR) at reducing cancer cell
viability in HCT116 colorectal cancer cells. Increasing
concentrations of FUdR between 0.5 and 5 .mu.mol/L demonstrated
dose-dependent decreases in colonies formed (FIG. 11A). Increasing
concentrations of PCI 10213, 10585 or 10586 between 0.78 and 12.5
.mu.mol/L had no significant effects on the number of colonies
formed (FIG. 11B).
[0357] To evaluate the combined drug effect, increasing
concentrations of PCI 10213, 01585 and 10586 were combined with a
fixed dose of 0.5 .mu.mol/L FUdR. Of note, 0.5 .mu.mol/L FUdR had
no significant effect on number of colonies formed compared to
vehicle-treated controls (FIG. 11A). PCI 10213, demonstrated
significant reductions in colonies formed when combined with FUdR
at concentrations of 6.25 .mu.mol/L and greater. However, all
concentrations of PCI 10286 combined with 0.5 .mu.mol/L FUdR
demonstrated reductions in colonies formed even at the lowest dose
of 0.78 .mu.mol/L when compared to the corresponding single agent
concentrations of PCI 10586 and 0.5 .mu.mol/L FUdR. Importantly,
PCI 10585 demonstrated no reductions in colony formation when
combined with FUdR at any concentration up to 6.25 .mu.mol/L with
modest reductions at 12.5 .mu.mol/L (FIG. 11C). These data support
the in vitro dUTPase inhibitor screen demonstrating that PCI 10586
has significantly more cell-based activity than PCI 10213 and that
PCI 10585 is significantly less potent than either 10213 or
10586.
[0358] Reference compound PCI 10950:
##STR00070##
demonstrated strong synergy when combined with fixed doses of FUdR
in all cell lines examined. Subsequently, increasing concentrations
of PCI 102951 and PCI 10952 were combined with a fixed dose of 0.5
.mu.mol/L FUdR to evaluate the combined drug effect. Substantial
reductions in colonies formed were observed when PCI 10951 was
combined with 0.5 .mu.mol/L FUdR compared to the corresponding
single agent concentrations of PCI alone or 0.5 .mu.mol/L FUdR. PCI
10952 demonstrated modest reductions in colonies formed when
combined with 0.5 .mu.mol/L FUdR in HCT116 cells under the
conditions tested.
[0359] Colony forming assays were subsequently performed to
evaluate the effectiveness of additional PCI compounds alone and in
combination with FUdR at reducing cancer cell viability in the
HCT-8 colorectal cell line model. FUdR at 1 .mu.mol/L demonstrated
no substantial effect on colonies formed. Increasing concentrations
of all PCI compounds between 1.56 and 6.25 .mu.mol/L also had no
significant effects on the number of colonies formed. Reference
compound PCI 10950 demonstrated strong synergy when combined with
the fixed dose of FUdR. Subsequently, increasing concentrations of
PCI compounds were combined with a fixed dose of 1 .mu.mol/L FUdR
to evaluate the combined drug effect. Substantial reductions in
colonies formed were observed when PCI 10951, PCI 10927, PCI 10928,
PCI 10929, PCI 10930, and PCI 10933 was combined with 1 .mu.mol/L
FUdR compared to the corresponding single agent concentrations in
HCT-8 cells.
[0360] Other compounds were also assayed employing the various
assays described herein, and can be assayed following these and
other assays known to the skilled artisan.
[0361] It should be understood that although the present invention
has been specifically disclosed by certain aspects, embodiments,
and optional features, modification, improvement and variation of
such aspects, embodiments, and optional features can be resorted to
by those skilled in the art, and that such modifications,
improvements and variations are considered to be within the scope
of this disclosure.
[0362] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. In addition, where features or aspects of the invention
are described in terms of Markush groups, those skilled in the art
will recognize that the invention is also thereby described in
terms of any individual member or subgroup of members of the
Markush group.
* * * * *
References